The objective of this work was to assess the influence of compressed propane treatment on the hydrolytic activity of three lipases (Amano PS, Amano AY30, and a non-commercial lipase from Yarrowia lipolytica) free, resuspended, and in immobilized form. To evaluate the effect of process variables on the lipase activity, a semifactorial experimental design with two levels and four variables was employed for free and immobilized lipases and a full 2 2 experimental design was carried out for lipases in solution. The residual activity was defined as the ratio of lipase activity before and after treatment with pressurized propane. For free and immobilized lipases, an enhancement in residual lipase activity in most of the experimental conditions investigated was observed. In the case of resuspended lipases, it is shown that enzyme kinetics is sensitive to treatment with compressed propane resulting in remarkable gains and losses of enzyme activity. In a general way, the results showed that the enzyme activity changes significantly depending on the enzyme, the presentation form, and the experimental conditions investigated, allowing the selection of operational conditions in terms of temperature, pressure, exposure time, and depressurization rate for advantageous application of these biocatalysts in hydrolysis reactions.