Abstract Isoniazid (IN), pyrazinamide (Pz) and rifampicin (Rf) are separated on YMC-ODS column. IN was derivatized with 2-fluorene-carboxaldehyde (FA). The separation was achieved using ethanol–chloroform–acetonitrile water by isocratic elution and detected at 337 nm. The detection limits were 0.11 ng, 0.2 ng and 13 ng/injection (5 μl) for IN, Pz and Rf, respectively. The method of analysis was applied to the pharmaceutical preparations and in the blood samples of the patients suffering from tuberculosis after undergoing chemotherapy with IN, Pz and Rf. The amounts quantitated in blood showed 0.97 to 1.58 μg/ml IN, 3.44 to 4.09 μg/ml Pz and 1.98 to 3.5 μg/ml Rf with coefficient of variations 0.8–1.8%, 0.9–1.3% and 0.8–2.1%, respectively.