From urine to serum: blood-based matrices reshape the future of anti-doping testing.

For more than five decades, urine has been the cornerstone biological matrix for anti-doping testing because of its noninvasive collection, broad analytical applicability, and favorable detection windows. However, evolving doping practices, including microdosing, increased use of biological medicines, and substances with limited urinary excretion, have exposed limitations of a urine-centered approach. At the same time, advances in liquid chromatography-tandem mass spectrometry (LC-MS/MS), high-resolution mass spectrometry (HRMS), and microsampling technologies have strengthened the potential of blood-based testing. Serum and dried blood spots (DBS) provide direct access to circulating analytes and physiological responses, offering complementary information to urine. These matrices have demonstrated particular value for detecting erythropoiesis-stimulating agents (ESAs), steroid esters, peptide hormones, and biomarkers incorporated into the Athlete Biological Passport (ABP). Emerging capillary blood collection devices further simplify sample collection and may facilitate wider implementation of blood-based anti-doping strategies. This review discusses the expanding role of serum, DBS, and capillary blood collection technologies in anti-doping analysis. Using ESA detection as an illustrative example, we examine how matrix selection affects detection capability and analytical performance. We also highlight applications in steroid monitoring, biological profiling, and future omics-based approaches, supporting the integration of blood-derived matrices into complementary multi-matrix anti-doping testing strategies.