Platelets have exhibited capabilities beyond clotting in recent years. Most of their functions are related to the nature of platelet adhesion. Establishing a facile method to understand the platelet adhesion and assess the platelet function through the mechanism and mechanics of adhesion is highly desired. Here, we report a generally applicable UV lithography technique with a photomask, which performs selective surface functionalization on large substrate areas, for creating stable, physical adhesive sites in the range of 12 μm to 3 μm. Our study demonstrated that the patterned surface facilitated probing of single platelet adhesion in a quantitative manner, and rendered platelets sensitive to adhesive proteins even at a low protein concentration. In addition, the platelet function in the presence of antiplatelet (anticancer) agents on platelets could be accurately estimated based on single platelet adhesion (SPA). This work paves a new way to understand and assess the blood platelet function. The SPA assay methodology has the potential to enable a rapid, accurate point-of-care platform suitable for evaluation of platelet function, detection of dysfunctional platelets, and assay of drug effects on platelets in cancer patients.