Tuipulotu, who scored a try after he started in the centres in game one in Brisbane, was robbed of a fairytale homecoming when he left out of match-day 23 as the Lions try to wrap up the series at the MCG on Saturday.
A Melbourne schoolboy before shifting to Scotland where he captains the national side, Tuipulotu was ruled out with a tight hamstring with Ireland's Bundee Aki named at 12.
Coach Andy Farrell said the 28-year-old, who qualified for Scotland through his grandmother, was shattered to miss out on playing in front of 90,000 in Melbourne.
"When you've got a little bit of a tight hamstring, it influences how you're possibly going to go, so we made the call to go with Bundee," Farrell said.
"100 per cent ( Tuipulotu) is absolutely gutted. He also knows that that's rugby, fairytales are not always written.
"Sione is a team man anyway. And it's the same with anyone who's missed out. Every single one of them has been rowing forward by putting the team first."
Four days after captaining the Lions in the midweek win over the First Nations and Pasifika, veteran playmaker Farrell, the son of the coach, will make his first Test appearance since the 2023 World Cup after being named on the bench.
Farrell made three changes to the starting line-up that won the first Test 27-19, with lock Joe McCarthy unable to overcome a foot injury and Olli Chessum the new face in the second row.
Andrew Porter will start at loosehead ahead of Ellis Genge, forming an all-Irish front row.
Aki will partner Scotsman Huw Jones in the midfield after Garry Ringrose was a last-minute withdrawal.
Farrell was set to go with the Ireland centre pairing after concussion symptoms that ruled Ringrose out of the opening Test returned at their Thursday training session.
"Garry was actually selected and unfortunately after training he's had to pull out," Farrell said of Ringrose, who managed 64 minutes in their midweek match.
"It's head-related again. It was literally as I was walking off the field. He came to me, and once that's mentioned, that's that.
"There was no incident. He was feeling good well in advance of Tuesday. He was fit and ready to play."
"It's unfortunate for Garry but we've always said it's about the squad, and nothing but about the squad."
Coach Farrell said they were expecting a "massive reaction" from the Wallabies after their Suncorp Stadium disappointment.
"We have put ourselves in a good position after the first Test, but we know there will be a massive reaction from this Wallaby team," Farrell said.
"Everyone saw the quality they have in Brisbane and we know we will have to be a lot better than we were last week."
BRITISH AND IRISH LIONS SQUAD TO FACE AUSTRALIA:
Andrew Porter, Dan Sheehan, Tadhg Furlong, Maro Itoje, Ollie Chessum, Tadhg Beirne, Tom Curry, Jack Conan, Jamison Gibson-Park, Finn Russell, James Lowe, Bundee Aki, Huw Jones, Tommy Freeman, Hugo Keenan. Bench: Ronan Kelleher, Ellis Genge, Will Stuart, James Ryan, Jac Morgan, Alex Mitchell, Owen Farrell, Blair Kinghorn.
