Von Bertouch suffered the injury during the third quarter of the loss to Western Bulldogs, but managed to play out the game.
A spate of players also went down with ACL injuries during the pre-season, with the problem a growing concern for the fledgling league.
Adelaide Crows co-captain Chelsea Randall was among the pre-season victims.
Individual clubs have tried hard to implement programs to strengthen the knees and surrounding areas of AFLW players, but the issue continues to be a major problem.
Fremantle’s AFLW high performance manager Kate Starre said Cain will undergo surgery in the coming week.
“It’s heartbreaking to have an injury to that extent and to have another ACL,” Starre said.
“We’ll go through the rehab process and get her up and about as soon as we can.
“We’ve seen with the ACL injuries that we’ve had so far that the girls have all responded so well to their rehab programs.
“Steph will be no different, she’ll be up and about as soon as possible.”
West Coast’s head of female football Adam Selwood said the club would do whatever it takes to help Hill recover and get back out on the field in 2021.
“It’s absolutely devastating for Ashton and the club,” Selwood said.
“We know how hard she has worked to get to this point to become a really well- prepared and consistent performer at AFLW level.”
Selwood also confirmed that defender Brianna Green would be unavailable indefinitely as she works her way back from general soreness.
Fremantle will start as hot favourites when they take on West Coast in the first ever AFLW western derby on Saturday.
A crowd in excess of 30,000 is expected to attend the match at Optus Stadium.