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Advances in cellular therapies have led to the development of new approaches for cell product purification and formulation, e.g., utilizing cell endogenous properties such as size and deformability as a basis for separation from potentially harmful undesirable by-products. However, commonly used additives such as Pluronic F-68 and other poloxamer macromolecules can change the mechanical properties of cells and consequently alter their processing. In this paper, we quantified the short-term effect of Pluronic F-68 on the mechanotype of three different cell types (Jurkat cells, red blood cells, and human embryonic kidney cells) using real-time deformability cytometry. The impact of the additive concentration was assessed in terms of cell size and deformability. We observed that cells respond progressively to the presence of Pluronic F-68 within first 3 h of incubation and become significantly stiffer (p-value < 0.001) in comparison to a serum-free control and a control containing serum. We also observed that the short-term response manifested as cell stiffening is true (p-value < 0.001) for the concentration reaching 1% (w/v) of the poloxamer additive in tested buffers. Additionally, using flow cytometry, we assessed that changes in cell deformability triggered by addition of Pluronic F-68 are not accompanied by size or viability alterations.