Inosine monophosphate dehydrogenase (IMPDH) is a crucial enzyme involved in the de novo synthesis of purine nucleotides. IMPDH activity is used to evaluate the pharmacodynamics/pharmacokinetics of immunosuppressant drugs such as mycophenolic acid and thiopurines. These drugs are often used to prevent organ transplant rejection and as steroid-sparing agents in autoinflammatory diseases such as inflammatory bowel disease and rheumatoid arthritis. Numerous analytical techniques have been employed to evaluate IMPDH activity in biological matrices. However, hyphenated LC techniques were most widely used in the literature. This review focuses on hyphenated LC methods used to measure IMPDH activity and provides detailed insight into the sample preparation techniques, chromatographic conditions, enzymatic assay conditions, detectors and normalization factors employed in those methods.