A sensitive, selective, and simple gas chromatography-mass spectrometry method is developed for quantitation of phenazopyridine (PAP) in human plasma using internal standard (diazepam). PAP and IS are extracted from plasma by liquid-liquid extraction and analyzed on a DB-5MS column with mass selective detector. Excellent linearity is found between 5-500 ng/mL (r = 0.9992, n = 7) for PAP in human plasma. The limit of detection is 0.3 ng/mL. Intra- and Inter-day precisions expressed as the relative standard deviation for the method are 1.37-6.69% and 1.24-6.01%, respectively. Extraction efficiency is more than 90%, and recoveries are in the range of 92.65-96.21%. This method is successfully applied for the pharmacokinetics and bioequivalence of 2 formulations of PAP in 18 healthy male volunteers who received a single 200 mg dose of each formulation.