Cell detachment is a procedure routinely performed in cell culture and a necessary step in many biochemical assays including the determination of oxygen consumption rates (OCR) in vitro. In vivo, cell detachment has been shown to exert profound metabolic influences notably in cancer but also in other pathologies, such as retinal detachment for example. In the present study, we developed and validated a new technique combining electron paramagnetic resonance (EPR) oximetry and the use of cytodex 1 and collagen-coated cytodex 3 dextran microbeads, which allowed the unprecedented comparison of the OCR of adherent and detached cells with high sensitivity. Hence, we demonstrated that both B16F10 melanoma cells and human umbilical vein endothelial cells (HUVEC) experience strong OCR decrease upon trypsin or collagenase treatments. The reduction of cell oxygen consumption was more pronounced with a trypsin compared to a collagenase treatment. Cells remaining in suspension also encounter a marked intracellular ATP depletion and an increase in the lactate production/glucose uptake ratio. These findings highlight the important influence exerted by cell adhesion/detachment on cell respiration, which can be probed with the unprecedented experimental assay that was developed and validated in this study.