In this study, three simple and rapid methods were developed for determination of some anticancer drugs Gemcitabine (GMB) and Etoposide (ETP) simultaneously in their pharmaceutical formulation. The first derivative amplitudes at 264 nm and 302 nm were selected for the assay of GMB and ETP, respectively. Calibration curves were established at 5 µg/mL-20 µg/mL for GMB and 2 µg/mL-15 µg/mL. The LOD of 0.89 µg/mL and 0.75 µg/mL, LOQ of 2.97 µg/mL and 1.42 µg/mL for GMB and ETP, respectively. The ratio derivative at 270 nm and 238 nm, calibration curves were established at 5 µg/mL–20 µg/mL and 2 µg/mL–15 µg/mL, LOD of 0.66 µg/mL and 0.25 µg/mL, LOQ of 2.19 µg/mL and 0.88 µg/mL for GMB and ETP, respectively. In the RP-HPLC method separation, the detection wavelength were 273 nm and 246 nm, the retention time was found to be 1.713 min and 5.021 min, linearity ranges of 0.5 µg/mL–35 µg/mL and 1 µg/mL-20 µg/mL, LOD of 0.09 µg/mL and 0.21 µg/mL and LOQ of 0.31 µg/mL and 0.80 µg/mL for GMB and ETP, respectively. The methods were applied for pharmaceutical formulation and were validated according to the procedure described in ICH guidelines.