Platelets play a role in both the innate and adaptive immune systems. Methods for detecting activated platelets and leukocyte-platelet aggregates (LPAs) are useful for basic and applied research concerning the role of platelets in inflammation and immune disorders. The aim of the study was to develop flow cytometric assays for detection of platelets binding to monocytes and neutrophils and for activated platelets in canine whole blood and to investigate the effect of physiologic agonists. Citrate anticoagulated whole blood was incubated with monoclonal antibodies against CD14 and CD61 for detection of LPAs, and the effect of various agonists was investigated. For detection of activated platelets, whole blood was incubated with monoclonal antibodies against CD62P and against a receptor-induced binding site on fibrinogen (CAP1) with CD61 as a platelet identifier. Isotype controls were prepared in parallel. The individual physiologic agonists ADP, collagen and epinephrine increased LPAs, CD62P and CAP1 binding only modestly. However, combinations of agonists gave more substantial increases. A dose-response relationship was seen using alpha- and gamma-thrombin, and ADP as agonists. In conclusion, we have developed flow cytometry assays to measure LPAs and platelet activation in canine whole blood, and have explored the effect of various physiologic agonists at different concentrations.