A method was developed to characterize the adhesion properties of single cells byusing protein‐functionalized atomic force microscopy (AFM) probes. The quantifica-tion by force spectroscopy of the mean detachment force between cells and agelatin‐functionalized colloidal tip reveals differences in cell adhesion properties thatare not within reach of a traditional bulk technique, the washing assay. In this lattermethod, experiments yield semiquantitative and average adhesion properties of alarge population of cells. They are also limited to stringent conditions and cannothighlight disparities in adhesion in the subset of adherent cells. In contrast, thisAFM‐based method allows for a reproducible and quantitative investigation of theadhesive properties of individual cells in common cell culture conditions and allowsfor the detection of adhesive subpopulations of cells. These characteristics meet thecritical requirements of many fields, such as the study of cancer cell migratory abilities.