The 32-year-old sustained the problem during the Gunners' 2-0 Champions League quarter-final second-leg win over Bayern Munich on March 29.
Little was brought off after just 12 minutes at the Emirates and subsequently missed Arsenal's 2-1 win over Manchester City on April 2 before the international break.
Jonas Eidevall refused to speculate on the severity of Little's injury immediately after the game, but the club have now announced that the former Scotland international will not play again this season.
The midfielder's problem is not expected to require surgery, though, and she is on course to return for pre-season training ahead of the 2023-24 campaign.
A medical update from Arsenal read: "We can confirm that Kim Little will miss the rest of the season with a hamstring injury.
"Kim sustained the injury in our UEFA Women's Champions League quarter-final win over Bayern Munich on 29 March and tests have subsequently confirmed that the injury will keep her sidelined for the remainder of the campaign.
"Our captain has already begun her rehabilitation and this process is progressing well. We expect her to be available for the start of preparations for the 2023-24 season.
"Our medical team will support Kim closely over the coming months and we look forward to see her back on the field of play soon."
Little ends the 2022-23 season with five goals and two assists under her belt from 16 appearances in the WSL and Women's Champions League, and her lengthy layoff is another damaging blow for Eidevall in the business end of the season.
The Gunners boss is already working without star forwards Beth Mead and Vivianne Miedema, both of whom are recovering from anterior cruciate ligament ruptures and will not feature for the remainder of the campaign.
Miedema has already ruled out her chances of playing for the Netherlands at this year's World Cup, while England boss Sarina Wiegman admitted that Mead would need a "miracle" to recover in time for the summer tournament.
Arsenal - who have not won a WSL title since the 2018-19 campaign - currently sit third in the table with 38 points from 16 matches, behind reigning champions Chelsea and leaders Manchester United.
Eidevall's side are three points worse off than Man United with a game in hand, and they travel to the league leaders on Wednesday in a crucial top-of-the-table showdown.
Following Wednesday's fixture, Arsenal head to Germany for the first leg of their Champions League semi-final with Wolfsburg on April 23 before the return fixture at the Emirates on May 1.