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The Giallorossi will face the Czech side in a two-legged affair to decide who will progress in the competition, with the second leg set to take place at Stadio Tre Fontane.
It will be the first time the ground has hosted European football in the women's game.
Alessandro Spugna's side beat Glasgow City and Paris FC to reach stage of this competition.
The two matches against Sparta will take place across September 20/21 and September 27/28.
"At this point in the competition any opponent would have been difficult but, given the other teams we could have faced, Sparta Prague are undoubtedly the one we know least about - even if a lot of their players are regulars for the Czech national team," Spugna said.
"At this point in the tournament any opponent will pose a major threat and both matches will surely be of the very highest level.
"For us it will be more great experience at continental level but we want to try and stay in the competition for as long as we can.
"The target is to give everything we have to reach the group stage - that would be an impressive achievement."
The Giallorosse began their league campaign at the weekend, beating Pomigliano 2-0 away from home.
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