Physiological anticoagulants are essential regulators of coagulation, and their deficiency shifts hemostasis toward a prothrombotic state, increasing the risk of venous thromboembolism. The main physiological anticoagulants, antithrombin (AT), protein C (PC), and protein S (PS), act via distinct mechanisms: AT directly inhibits active proteases, mainly thrombin and factor Xa, while PC deactivates activated factors V and VIII with PS as its cofactor. Deficiencies may be inherited (due to genetic mutations) or acquired (resulting from reduced synthesis or increased consumption). Laboratory diagnosis relies on functional and antigenic assays, although results can be influenced by anticoagulant therapy and clinical context. First-line investigations commonly involve the use of chromogenic assays for AT and PC activity, while PS assessment typically begins with free antigen measurement. Diagnostic challenges include assay limitations, biological variability, and treatment interference. Although genetic testing may overcome some limitations, it remains incompletely accessible. Advances in multigene thrombophilia panels are likely to support a more personalized diagnostic approach in the future.