Platelets are the smallest and perhaps the most versatile components of human blood. Besides their role in coagulation and the maintenance of vascular integrity, they are involved in many physiological processes, ranging from immune response and leukocyte recruitment to the production of antimicrobial peptides and immune-suppressive factors like TGF-β. These versatile abilities make platelets interesting for researchers from different disciplines. However, beside profound investigation into platelets’ physiological role, there is a need for correct, standardized and thus reproducible quantification of platelet parameters. Mean platelet volume (MPV) is a widespread prognostic marker for several conditions, such as, acute coronary syndrome, chronic kidney disease and liver cirrhosis. Platelet activation is regarded as a marker for inflammatory processes, for example in autoimmune diseases such as type-1 diabetes, systemic lupus erythematosus and rheumatoid arthritis. The monitoring of platelet function is relevant for patients receiving antiplatelet medication. Platelet parameter measurement is affected by the choice of in vitro anticoagulant, the measurement technology and the time delay after sampling. This review focuses on the pre-analytical variability that arises as a result of the use of different in vitro anticoagulants and analyzer technologies when determining platelet parameters, since, even approximately 180 years after the discovery of platelets, there is still no standardized procedure.