The Edinburgh club secured a 2-0 victory to silence Accies fans on Wednesday and now play host at Easter Road, where they are highly fancied to hold off the Division One club and preserve their Scottish Premiership status.
Midfielder Kevin Thomson will be pushing for a start having been named on the bench for the first leg, while defender Paul Hanlon remains unavailable due to injury.
Accies manager Alex Neil left himself on the substitutes' bench for the clash at New Douglas Park and he was joined there by well-travelled forward Jason Scotland, who will be hoping to start in the capital.
Hibs striker Paul Heffernan said ahead of the clash: "We know there's still a long way to go. It's nice to have a 2-0 lead, but we definitely won't take our foot off the pedal.
"Hamilton are a good side. We're definitely not going to be complacent and we know we've got a massive challenge ahead still.
"It's the last chance of the season for both teams; to stay in the Premiership for ourselves or for them to come up.
"I'm sure they'll throw everything at us and we'll be very prepared for that and hopefully they'll leave a few gaps and we can exploit them ourselves."
Accies goalkeeper Kevin Cuthbert added: "Like any cup tie, form can go out the window, so we haven't spoken much about Hibs' recent results.
"If you went with the form book, most people would have expected us to win the other night, but Hibs did. We're the ones going for promotion rather than having to stave off relegation.
"We know we can go all out for the win because we have nothing to lose. If we don't get the result, we will be disappointed, but nothing will change for this club.
"The team from the Premiership, though, has a lot at stake if they don't win. Hopefully, if we can get an early chance, we can take it and crank up the pressure on Hibs."