To control their attachment to substrates and other cells, cells regulate their adhesion receptors. One regulatory process is receptor crosstalk, where the binding of one type of cell adhesion molecule influences the activity of another type. To identify such crosstalk and gain insight into their mechanisms, we developed the stimulated single-cell force spectroscopy assay. In this assay, the influence of a cells adhesion to one substrate on the strength of its adhesion to a second substrate is examined. The assay quantifies the adhesion of the cell and the contributions of specific adhesion receptors. This allows mechanisms by which the adhesion is regulated to be determined. Using the assay we identified crosstalk between collagen-binding integrin α ₁β₁ and fibronectin-binding integrin α₅β₁ in HeLa cells. This crosstalk was unidirectional, from integrin α₅β₁ to integrin α₅β₁, and functioned by regulating the endocytosis of integrin α₅β₁. The single-cell assay should be expandable for the screening and quantification of crosstalk between various cell adhesion molecules and other cell surface receptors.