Winks, who has this season become a key man for his club and worked his way into the national team, last played in the first leg of Spurs' Champions League quarter-final against Manchester City at the start of the month.
The 23-year-old has been carrying an injury for some time and manager Mauricio Pochettino was last week unsure of his participation in the semi-final meeting with Ajax.
"We don't know because it is a problem that is one day very good, next day not very good," Pochettino said of Winks. "It is about assessing each day."
But it seems highly unlikely that Winks will feature again this season, with the first leg against Ajax on Tuesday and the second leg to come next week.
Winks underwent surgery on Monday and a short statement on Tottenham's Twitter page added: "[Winks] will be monitored by our medical staff as he continues his recovery before commencing rehabilitation."
@HarryWinks has today undergone groin surgery.— Tottenham Hotspur (@SpursOfficial) April 29, 2019
The @England midfielder will be monitored by our medical staff as he continues his recovery before commencing rehabilitation. #COYS pic.twitter.com/oaJtqvaRc3
Spurs had earlier confirmed that Winks would miss the midweek game at Tottenham Hotspur Stadium.
The midfielder has made 26 Premier League appearances this season, along with nine in the Champions League.
Winks has just three England caps but played in a 3-2 win over Spain last year before injuries kept him out of subsequent matches.
Mauricio Pochettino has a growing injury list to manage as his side look to advance to the Champions League final while simultaneously securing their qualification to next year’s competition.
Star striker Harry Kane is out with an ankle complaint, though his manager recently expressed hopes the England captain would be available for the final if Spurs make it.
Erik Lamela and Serge Aurier are out with long-term injuries as well.
Son Heung Min is out for the first leg against Ajax through suspension with Lucas Moura expected to lead the line against the Dutch club.
Tottenham were handed a boost this week as Moussa Sissoko returned to training after missing three games with his own groin problem.
Sissoko now appear likely to be able to contribute in some way on Tuesday.