Humza Yousaf took a swipe at SNP leadership rival Kate Forbes over her opposition to same sex marriage during the first party hustings.
Yousaf said gay Scots would have to look the next First Minister in the eye and know they would advance their rights.
Three candidates - Yousaf, Forbes and Ash Regan - went head to head in Cumbernauld in the contest to succeed Nicola Sturgeon.
The race has so far been dominated by Forbes, an evangelical Christian, saying she would not have voted for same sex marriage in 2014.
After a question on whether someone of faith can rise to the top, Health Secretary Yousaf mentioned gay rights without the issue being raised.
He said: “It's so, so important that whoever is First Minister, that whether you're lesbian, gay, bisexual, transgender, whoever you are, you can look the First Minister in the eye, and you can have confidence that they will not only protect your rights, that they will not only tolerate you, but that they will celebrate you and that they will advance your rights where they possibly can.”
Forbes, the Finance Secretary, said: “I've answered more questions on faith in a matter of days than most politicians will ever do in their lifetime. Obviously, from my perspective, the last few weeks I've learned a lot. I recognise that there are things that I have phrased or framed that I could have done better [at], and I've been very clear about that last week.
“I do hope that in Scotland our pluralistic and tolerant society can allow for people of faith to rise to the highest office. I hope too that we should hold our politicians to that high bar of being honest and open, so we know who they are, and can then make an informed decision on that basis.”
Regan answered by saying she had been “upset” by the way Forbes had been treated by the media, saying: “I absolutely think it should be possible for people to rise to the top, even if they have a strong faith.”
Another flashpoint was on what the candidates would do after the UK Government blocked the Scottish Government’s gender recognition reforms.
The legislation is aimed at making it easier for trans people to change their gender, but critics believe it could put women and girls at risk.
Yousaf committed to going to court to challenge the UK Government’s Section 35 order, saying: “Let me make sure that you are hearing me absolutely clearly. We must challenge section 35. We absolutely have to.
“This is about the principle of our democracy. If we cave in, if we roll over to a Westminster power grab, they will come after legislation after legislation after legislation. They are already threatening to do it, time and time and time again.
“What kind of message does that send to the Scottish people that we're not willing to stand up for devolution?”
Forbes, who is opposed to the legislation and has said she would not go to court, said: “I think we need to act like we are in many cases an independent government, an independent country already.
“I would seek legal advice, but I'd far rather sort out the legislation ourselves without having to go to court or without having to seek Westminster's permission around what we do next.”
Regan, who quit Sturgeon’s administration in protest over the gender reforms, said the Government had “really lost the trust of the country” over the issue.
She said: “I wouldn't challenge the section 35, not because I think that it's okay for the UK to challenge our legislation, but for the simple reason that we're going to lose that court case.
“And we are going to be throwing probably hundreds of thousands of pounds of public money into something that the public don't support.”
The candidates also outlined their approach to how they would pursue independence.
Forbes said: "If elected as First Minister, the first job I would do on day one is to set out an independence action plan, a plan which outlines precisely how we are going to increase support for independence, but more than that.
“It sets out that in 2024 at the next Westminster election, we will put independence front and centre. And in that election, we will fight for the right to hold a referendum within three months of that election.”
Yousaf said: “We can’t get bogged down by process, we have to talk about our vision for independence.
“Because you know what, if we build the popular support for our independence, if we ensure that we get a sustained majority for independence, not fluctuate from poll to poll, but a sustained majority, then independence will become the political reality, the political inevitability. But we have to get out there and talk to people.”
Regan said: “We need good governance to achieve our goals, we need to restore the trust of the Scottish people in our ability to govern wisely, to govern sensibly and to govern competently.
“And I believe in a very collegiate style of governance where people of ability are appointed to the top positions and where they are allowed the freedom to flourish and get on with the job in their own portfolio.”
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