Adam Lallana’s side secured a home play-off tie by finishing in the top eight places and will welcome 11th-place Aston Villa to the south coast.
A date has not yet been confirmed for the match, which could take place at St Mary’s, Eastleigh‘s Silverlake Stadium or Staplewood Campus as soon as next week.
Saints finished the 20-game PL2 regular season with 36 points to their name, winning 10 games, drawing six and losing four.
Two of those defeats came at the end of the campaign, with second-place Manchester United beating them 2-1 and Stoke earning a 1-0 win.
Top goalscorer Nick Oyekunle, who ended the season with 10 goals, saw red in that defeat to Stoke. Saints did not face Aston Villa during the regular season.
Southampton reached the PL2 play-off final last season, losing 2-0 to Manchester City at the Joie Stadium.