Protein C (PC) and protein S (PS) are vitamin K-dependent glycoproteins, that act as natural anticoagulants. The proteolytic activation of PC by thrombin occurs on the surface of endothelial cells and involves thrombomodulin and endothelial PC receptor. In the presence of PS, phospholipids and calcium, activated PC (APC) inactivates membrane bound factors V (FVa) and FVIIIa by their cleavage at the specific arginine residues. PC and PS deficiencies are inherited as autosomal dominant disorders associated with recurrent venous thromboembolism (VTE) and, in most cases, derived from heterozygous missense mutations (78% and 63%, respectively). Heterozygous PC deficiency is found in 6% of families with inherited thrombophilia, in 3% of patients with a first-time deep vein thrombosis (DVT) and 0.2-0.3% of healthy individuals. The PS deficiency is detected more commonly than PC deficiency and its prevalence has been estimated with a less than 0.5% in the general European population and 2% to 12% of selected groups of thrombophilic patients. Approximately 75% of PC-deficient patients have type I deficiency and 95% of PS-deficient patients develop type I and type III of PS deficiency. The diagnosis of PC and PS deficiencies is challenging, many preanalytical and analytical factors may affect the PC/PS levels. Molecular analysis of the PC and PS genes (PROC and PROS1, respectively) involves direct gene sequencing and if negative, multiplex ligation-dependent probe amplification (MLPA) method. Patients with low PC and PS levels and the known mutation within PROC or PROS1 genes combined with other genetic or environmental thrombosis factors are at increased risk of recurrent thromboembolic events and require lifelong oral anticoagulation.