In this study, a sensitive, simple, and selective reversed HPLC-UV method has been developed for the quantitation of nitrofurantoin (NFT) and phenazopyridine (PHZ) in human urine samples. The target analytes were quantified using a gradient elution mobile phase consisting of 5 mmol/L sodium dihydrogen phosphate, pH 3 (A), and acetonitrile (B) at a flow rate of 1 mL/min and UV detection at 370 nm. Separation of NFT and PHZ was achieved using a reversed-phase C18 column (150 x 4.6 mm (i.d.), 5 µm particle size). The method was validated according to ICH guidelines and the calibration regression analysis for the calibration plot of PAP showed a good linear relationship over the concentration range of 0.5-40 μg/mL and 0.2-20 μg/mL for PHZ and NFT, respectively. The limit of detection (LOD) was 0.01 μg/mL for both PHZ and NFT, while the limit quantitation (LOQ) was 0.025 and 0.04 for PHZ and NFT, respectively. Intra- and Inter-day precisions calculated as the relative standard deviation %RSD values of the intra-day and inter-day were ≤5.5% at three concentration levels of the two analytes. the recovery expressed as percent relative error %RE ranged from -2.5% to 6%. The proposed method was applied for the analysis of volunteer human urine samples by the dilute and shoot technique.