CD40-CD40L interactions have been involved in inflammation and thrombosis. Several diseases are characterized by inflammation, hypercoagulability and increased prevalence of thromboembolic events. In the past decade, a series of preclinical and clinical studies has provided more insight into the pathogenetic mechanisms linking inflammatory mediators to the activation and regulation of the haemostatic system. In particular, the study of CD40-CD40L interactions has greatly contributed to understanding the role of platelets in a variety of pathophysiological conditions, including atherothrombosis, immunoinflammatory diseases and, possibly, cancer. A wide variety of preclinical and clinical studies have generated clinical interest in the use of CD40L as a prognostic marker of thrombotic risk. However, the use of sCD40L in clinical studies requires reliable methods. For the correct interpretation of results, clinical and research laboratories and physicians must be aware of the limitations of immunoassays for this cytokine, which underlines the need for standardization of preanalytic conditions. This review will focus on biochemical evidence of CD40L involvement in platelet activation, contribution of platelet-derived CD40L to inflammation, thrombosis and neoangiogenesis, and possible methodological pitfalls regarding the appropriate specimen and preparation for laboratory evaluation of blood soluble CD40L as a biomarker in various human diseases characterized by underlying inflammation, such as atherothrombosis, cancer and immuno-inflammatory diseases.