Following a goalless first half, the Saints took control of the tie and took the lead through Danny Ings before Stuart Armstrong sealed the victory in injury-time.
The victory is their first win in their previous five games, whilst Wolves suffer their first defeat after two solid results against Leicester City and Arsenal.
Story of the match
Following Sunday's goalless draw with Leicester City, Nuno Espirito Santo shocked a number of Wolves fans' by making six changes that included Adama Traore, Pedro Neto and Conor Coady all dropping down to the bench.
For Southampton, who conserved many regular starters in their starting line-up, started the better of the two teams, with Armstrong, Moussa Djenepo and Ings trying their luck in the opening encounters of the tie.
The hosts really struggled to get into the game, with Fabio Silva, Vitinha and Morgan Gibbs-White all struggling to control any aspect of the game when Wolves did manage to counter.
After a pretty uneventful first half, it took Ralph Hssenhuttl's side only five minutes to open the scoring in the second half as Ings' effort deflected off Ruddy onto himself to sneak into the bottom corner.
The goal is the striker's eighth of the season and only his third ever career goal against the Wanderers.
Wolves almost responded immediately after falling behind when their high press won the ball back from the Saints defence before Ki-Jana Hoever crossed for Silva to volley just over.
Nathan Redmond had the games next big chance with 25 minutes remaining after some impressive play from the visitors, but Ruddy was quick to deny the winger of his side falling two goals behind.
Espirito Santo introduced Willian Jose, Traore and Neto to try and pin Southampton back and attempt to force extra-time, but all three, like the rest of the Wolves team, struggled to get into the game in the freezing conditions.
However, the home side recorded their first shot on target at Molineux for the first time in over three hours of football when Traore should have scored from six-yards out. Neto's low cross was missed by Mohammed Salisu to reach Traore, but Fraser Forster managed to scramble across his line to deny the pacy winger.
Southampton's passage to the quarter-final of the FA Cup was sealed in the 90th minute when Nathan Tella robbed Ruddy of the ball before cutting the ball back for Armstrong who superbly curled the ball off-the-post and into the net.
Wolves have only recorded two shots in over three hours of football at Molineux
Since Joao Moutinho's wonder strike to earn Wolves the 2-1 victory over Arsenal last week, one of the main concerns for Nuno going forward would be the lack of creativity and opportunities created by his side.
Tonight, they only managed three shots (one on target), whilst only creating six chances against Leicester on Sunday (one on target). With Raul Jimenez still out, Willian Jose is yet to find the net for the Wanderers, whilst Fabio Silva continued to struggle.
They are currently the fifth lowest-scoring team in the Premier League (the other four being the bottom four teams). Going forward, Wolves must start games quicker and force their opponents to defend more.
Following the game Nuno discussed the problems from the defeat in the cup, saying: "We didn't compete so well. We were organised but concede easy goals.
"[Lack of shots] is a big concern. We didn't produce enough. The ones that were good required better finishing."