The Italian side will have home advantage for the first leg, which will take place at the Stadio Olimpico in front of at least 35,000 supporters.
Match preview
Roma Women collected 13 points from their six group games to progress to finish in second place and one point behind German champions Wolfsburg.
Alessandro Spugna's side know that they will have to be at their very best to trouble one of the best sides Europe has to offer, but their promising domestic campaign suggests that Roma are capable of causing Barcelona a few problems.
Not only have they set up a Coppa Italia final against Juventus, but Roma are also on course to end the Turin club's five-year reign as Serie A champions.
Thet claimed their seventh consecutive league victory on Friday, hammering Fiorentina 5-1 to maintain their eight-point lead at the top of the table.
Including qualifying, Roma have won three and drawn one of their home Champions League games this season, which will provide them with a source of inspiration ahead of Tuesday's first leg.
Barcelona, meanwhile, went close to retaining the Champions League trophy they won in 2021 after they reached their third European final in four years.
However, a 3-1 defeat to Lyon in last season's final denied Barcelona a second Champions League crown, and they will look to use that disappointment as motivation for the knockout rounds.
Having won 22 league games this season, the Catalan giants are set to win their fourth consecutive league title, but they will be unable to defend their Copa de la Reina trophy after they were excluded from the completion for fielding an ineligible player.
Nevertheless, Jonathan Giraldez's side still have a chance to finish the season with two pieces of silverware, however, their first priority will be to progress to the Champions League semi-finals for a fifth consecutive season.
Having won their last six competitive away games by a combined 20-0 scoreline, Barcelona will travel to Italy with an air of invincibility.
Roma Women Women's Champions League form:
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Roma Women form (all competitions):
Barcelona Women Women's Champions League form:
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Barcelona Women form (all competitons):
Team News
Roma will have to cope without the services of Sophie Roman Haug, who has been sidelined since January with an ankle injury.
Former Barcelona players Andressa Alves and Vicky Losada will feature in the matchday squad, although the latter may have to settle for a place on the bench.
Having scored 19 goals in 33 competitive appearances this term, Valentina Giacinti will offer one of the main goal threats for the hosts.
As for the visitors, they are unable to call upon Alexia Putellas, Mariona Caldentey and Claudia Pina, who are sidelined with injury issues.
With the focus on Tuesday's Champions League encounter, a number of key players dropped to the bench for Friday's league win against Valencia, but the likes of Lucy Bronze, Mapi Leon, Patri Guijarro, Caroline Graham Hansen and Asisat Oshoala will all return to the starting lineup.
After coming off the bench to score a brace in the 5-1 win over Valencia, Salma Paralluelo will also be brought into the side for the midweek trip to the Italian capital.
Roma Women possible starting lineup:
Caesar; Bartoli, Wenninger, Linari, Minami; Andressa, Greggi, Giugliano; Serturini, Haavi, Giacinti
Barcelona Women possible starting lineup:
Panos; Bronze, Paredes, Leon, Rolfo; Bonmati, Walsh, Guijarro; Graham Hansen, Paralluelo, Oshoala