The order of speaking in the August 5 debate has been decided with a series of coin tosses, with the Scottish National Party leader speaking first in the opening statements and last in the closing statements.
Representatives from both sides of the campaign met at the debate venue, the Royal Conservatoire of Scotland, today to decide the running order, STV said.
Mr Darling will lead the cross-examination section, in which the two men will quiz each other on key referendum questions, following initial questioning by STV political editor Bernard Ponsonby.
This will be followed by questions from the 350-strong audience, which has been selected in conjunction with pollsters Ipsos-MORI Scotland to represent fairly Yes, No, and undecided voters.
Mr Salmond has repeatedly insisted he should debate with David Cameron over Scotland's future in the UK ahead of September's independence referendum, although the Prime Minister has consistently refused to do so.
Last month, a clash between the rival campaigns broke out over the timing of the debate.
Mr Salmond accepted an invitation from STV to take on Mr Darling on August 5 but the Better Together campaign said it was ''disappointed'' that the STV debate would not take place on July 16, as the broadcaster had originally planned.
The two men will finally debate at 8pm on Tuesday in a two hour session, which will conclude with reaction from both sides of the campaign in STV's "spin room". A special follow-up edition of Scotland Tonight will be broadcast at 10.30pm.
Gordon Macmillan, head of STV News, said: "We've had considerable media interest from across the UK and around the world in the first debate involving Alex Salmond and Alistair Darling. We anticipate the STV programme next Tuesday will be informative and engaging, and will be available to watch live on air and online."
Viewers from the rest of the UK and across the world can watch the debate online with a globally available livestream on the STV Player, with viewers encouraged to join the debate from home on Twitter, Facebook, and Google+.