Background: Modified Vaccinia Ankara (MVA) vectors are highly immunogenic vaccine platforms for the delivery of recombinant antigens. Efficient downstream processing is still challenging, particularly because substantial fractions of the virus remain intracellular. While chemical cell lysis that releases MVA particles into the supernatant before clarification can greatly enhance process efficiency and scalability, this step remains insufficiently characterized. Methods: This study assessed the compatibility of ionic, non-ionic, and zwitterionic detergents with the virus as purification target. Polysorbate 20 (Tween 20) was selected as a candidate detergent and evaluated across harvest times of 48–72 h post-infection (hpi) at concentrations of 0.01–0.5% (v/v). Results: The addition of 0.01% to 0.05% Tween 20 at 48 hpi resulted in a twofold increase in supernatant virus within one hour of application. Extended exposure to Tween 20, combined with a 650 mM mixture of NaCl, NaBr, and KCl, promoted virus particle release. However, Tween 20 concentrations above 0.1% reduced MVA infectivity. A filtration cascade using pore sizes of 5 µm and 1.2 µm achieved product yields of 77–83% at 48 hpi and 41–69% at 72 hpi, respectively. Host-cell DNA is an important contaminant during viral vector processing. However, the application of 0.05% (v/v) Tween 20 resulted in a 35% reduction of dsDNA released into the culture supernatant; the nuclei could not be preserved intact under high-salt conditions to avoid the release of cellular DNA. Conclusions: In summary, this comprehensive data demonstrated that non-ionic detergents can be used to induce cell lysis while maintaining infectious activity of enveloped MVA.