Thrombophilia, characterized by a predisposition to abnormal blood clot formation, is influenced by both genetic and acquired risk factors affecting coagulation mechanisms. This chapter provides a comprehensive overview of the laboratory tests critical for diagnosing thrombophilia, emphasizing recent advancements in diagnostic methodology. Inherited thrombophilias involving specific genetic mutations, such as Factor V Leiden and Prothrombin G20210A, and natural anticoagulant deficiencies contrast with acquired forms linked to environmental factors, including antiphospholipid syndrome. Essential laboratory tests, such as PCR for genetic mutations, functional assays for natural anticoagulant levels, and coagulation profiles, are highlighted. Novel technologies like next-generation sequencing enhance genetic diagnostic capabilities, while emerging biomarkers offer promise for better understanding antiphospholipid syndrome. Despite significant progress, challenges persist, particularly regarding the timing of testing, false-positive/negative results, and interpretation of results, necessitating expert involvement. Ultimately, accurate laboratory diagnosis is pivotal for guiding clinical decisions, including anticoagulation therapy and genetic counseling to reduce the risk of thrombosis.