Heterozygous deficiency of Protein S (PS) increases the risk for developing thrombosis. Many acquired conditions alter plasma PS levels. These complex interactions of PS in plasma make it imperative that clinical PS assay limitations are understood so that the assays are reliable, reproducible and specific to diagnose true genetic abnormalities based on plasma phenotype alone. Unfortunately, the diagnosis of PS deficiency is difficult and complicated. Three basic assays can be utilized for assessing PS in plasma: PS activity assay, Free PS antigen assay, and Total PS antigen assay. This article will review these clinical assays and their associated problems. We also discuss the confounding and interfering factors that make it difficult to obtain an accurate diagnosis of PS deficiency.