Activated protein C (APC) is an effective anticoagulant that inactivates activated factors V and VIII in the coagulation cascade. Deficiencies in protein C (PC) can increase the risk of thrombosis. Inherited PC deficiency can be classified as either quantitative (type I) or qualitative/functional (type II), with type II further subdivided into IIa and IIb. Additionally, PC deficiency can be acquired due to various conditions, such as liver disorders, infections, and the use of vitamin K antagonists. Beyond its anticoagulant properties, APC also plays important roles in fibrinolysis, immunoregulation, and cytoprotection. Clinical laboratories frequently use the chromogenic PC assay to identify deficiencies due to its high specificity; however, it may not detect type IIb deficiency. The clotting-based PC assay can identify type IIb deficiencies but has a notably lower specificity. The main aim of PC antigen assays is to differentiate between type I and II deficiencies when abnormalities in PC function are detected. Different factors can affect the measurement of PC activity and antigen levels. These factors can occur before, during, or after the testing process. Furthermore, researchers are currently evaluating the effectiveness of different recombinant APC variants. These variants do not have anticoagulant activity but still provide cytoprotective benefits. Some of these treatments are currently being tested in clinical trials. Consequently, developing a reliable method for quantifying circulating APC in plasma is of significant interest, and several proposed methods for measuring APC come with their advantages and disadvantages.