The hosts are unbeaten in six competitive games, while the visitors have lost just one of their previous nine matches.
Hertha are competing in the last 16 for the fourth time in six seasons after they eased through the opening two rounds of the Pokal.
They began their cup campaign with a dominant 5-0 victory over fourth-tier Carl Zeiss Jena, before recording a 3-0 win against Bundesliga club Mainz.
That commanding victory over top-flight opposition has helped Hertha embark on a six-game unbeaten run (W3, D3), where they have scored at least two goals in five of those matches.
After playing out three consecutive draws, Pal Dardai's side returned to winning ways with a resounding win against Elversberg on Sunday, with Florian Niederlechner netting a hat-trick in a 5-1 victory that leaves Hertha in eighth place and seven points adrift of third-placed Hamburg.
While they will be keen to catch Hamburg in the league, their current priority will be to get the better of die Rothosen in Wednesday's cup tie, as they dream of reaching the quarter-final stage for the first time since the 2015-16 campaign.
In contrast to their opponents, HSV have had to battle hard against two third-tier sides to secure their place in the last 16 for the second time in three seasons.
They claimed a 4-3 extra-time victory to squeeze through their first-round tie with Rot-Weiss Essen, before they held their nerve from the penalty spot to win a shootout against Arminia Bielefeld in their most recent cup outing.
Hamburg will head into Wednesday's contest off the back of an eventful derby clash with rivals St Pauli, where they fought back from two goals down to rescue a point thanks to second-half efforts from Robert Glatzel and Immanuel Pherai.
Although their promotion bid is their primary focus, Tim Walter's side will also be looking to produce a memorable cup run, just like in 2021-22 when they made their way to the semi-finals before falling to defeat against Freiburg.
HSV, who won the last of their three Pokal trophies in 1987, may be able to draw confidence from the fact that they have already beaten Hertha this season, cruising to a 3-0 home win when the two sides met back in August.
Experienced defender Toni Leistner is also a doubt for the midweek cup tie after he missed the win over Elversberg due to an injury issue.
Having scored five goals in his last three competitive appearances, Niederlechner will look to continue his rich vein of form when he lines up against Hamburg on Wednesday.
Bakery Jatta, who missed the derby through suspension, is expected to come back into the starting lineup for Wednesday's cup tie.
Hertha Berlin possible starting lineup:
Ernst; Kenny, Gechter, Kempf, Zeefuik; M. Dardai, Klemens; Winkler, Niederlechner, Reese; Tabakovic
Hamburger SV possible starting lineup:
Raab; Mikelbrencis, Ramos, Ambrosius, Muheim; Pherai, Meffert, Benes; Jatta, Dompe, Glatzel
We say: Hertha Berlin 2-2 Hamburger SV
Having been taken to extra time in their opening two cup ties, Hamburg may be bracing themselves for another close-fought contest.
While we believe that the visitors will be taken the distance, we think that they will hold their nerve to get the better of Hertha inside 120 minutes.
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