The reigning French champions have a slight advantage after coming from behind to claim a narrow 2-1 victory in Lisbon last week.
Match preview
Lyon, who have been crowned European champions on eight occasions, showcased all of their Champions League prowess to produce a fightback in the first leg against Benfica.
The French side trailed at the break following Andreia Faria's opener, but they managed to pull themselves level through Delphine Cascarino, before Sara Dabritz found the net to complete the turnaround.
Although Sonia Bompastor rotated her squad at the weekend, Lyon claimed another impressive result, cruising to a 7-0 away league win over Lille thanks to an Amel Majri hat-trick, a Vicki Becho and efforts from Dzsenifer Marozsan and Liana Joseph.
After recording a third consecutive victory, Lyon will hope to pick up another positive result in Wednesday's second leg, as they look to better last season's quarter-final exit to Chelsea.
The hosts will certainly fancy their chances of reaching the next round, having avoided defeat in all seven of their European outings this term (W5, D2).
Benfica have already made history in Europe this season, taking nine points from six group matches to become the first Portuguese team to reach the quarter-final stage.
However, their hopes of continuing their memorable journey were dented by last week's result, which represented their first home European defeat of the 2023-24 campaign.
Benfica quickly bounced back to ease to a 4-1 victory in Saturday's league meeting with Braga following a Kika Nazareth brace and goals from Faria and Marie-Yasmine Alidou.
The Portuguese champions will now head on their travels, where they have put together an 11-game unbeaten away run since losing 5-0 to Barcelona in November.
If they are to have any hope of progressing, Benfica will need to become the first away team to beat Lyon in regulation time since Chelsea recorded a 1-0 win in last season's quarter-finals.
Lyon Women Women's Champions League form:
W
W
D
W
D
W
Lyon Women form (all competitions):
Benfica Women Women's Champions League form:
W
W
D
D
D
L
Benfica Women form (all competitions):
Team News
Lyon are expected to be without Norwegian forward Ada Hegerberg, who has missed the last three matches through injury.
Griedge Mbock, Lindsey Horan, Dabritz and Cascarino are all expected to be recalled to the lineup after being rested at the weekend.
Wendie Renard is likely to remain on the bench despite returning from injury to play the final half-hour against Lille.
As for the visitors, Nycole Raysla is likely to miss out through injury after being forced off in the 15th minute of the first leg.
Benfica are also unable to call upon midfielder Leticia Almeida, who is continuing her rehabilitation from knee surgery.
Portugal international Jessica Silva is another fitness concern, having been absent from Benfica's last two matchday squads.
Lyon Women possible starting lineup:
Endler; Carpenter, Mbock, Gilles, Bacha; Egurrola, Horan, Dabritz; Majri, Cascarino, Diani
Benfica Women possible starting lineup:
Pauels; Lais, Costa, Ucheibe; Alves, Gasper, Faria, Falcon; Marie-Yasmine Alidou, Nazareth, Davidson