Two-dimensional cell cultures are established models in research for studying and perturbing cell-type specific functions. The simplicity of these models facilitates the reproducible manipulation of cultured cells. Limitations, however, relate to cell growth in a monolayer using standard cell culture media. Although such media are in use for decades, their formulations do not mimic the composition of the originating human cell environment in physiological or pathological conditions. In this study, we selected three frequently-used cancer cell lines SW620 (colon cancer), MCF7 (breast cancer), and A375 (melanoma) to determine the impact of a newly formulated human plasma-like medium (HPLM) on cell proliferation and mitochondrial function. Mitochondrial bioenergetic profiles were analyzed by high-resolution respirometry, revealing metabolic reprogramming in cells cultured in standard and HPLM media. Notably, the culturing of differentially proliferating cells in HPLM affected key mechanism of cellular respiration. All three cancer cell lines displayed an amending bioenergetic profile, particularly related to mitochondrial density and mild uncoupling of respiration. Importantly, only cells cultured in HPLM displayed mitochondrial dysfunction upon exposure with the clinically prescribed cancer drug and kinase inhibitor sunitinib. These findings highlight that cell culture media redirect mitochondrial function and affect cancer drug sensitivities. To advance the translational potential of cell culture models we propose to prioritize media with a human plasma-like composition for analyzing bioenergetic profiles and for determining target-oriented drug efficacies.