Two chromatographic methods have been developed for determination of gemifloxacin in human plasma with three co-administered drugs, theophylline, warfarin and omeprazole. First method depends on reverse phase high performance liquid chromatography. The plasma sample was extracted using acetonitrile. The method was linear over the concentration range 0.05 to 6 µg/mL, 0.25 to 8 µg/mL, 0.1 to 10 µg/mL and 0.1 to 6 µg/mL of gemifloxacin mesylate, theophylline, warfarin and omeprazole, respectively. The mobile phase used was prepared by mixing acetonitrile and 0.02 mol L-1 potassium dihydrogen phosphate buffer (pH adjusted to 2.5 using ortho phosphoric acid) in a ratio 10:90 with the addition of 1% TEA and flow rate 1 mL/min in isocratic mode and UV-detection at wavelength 254 nm. Second method depends on densitometric thin layer chromatography. The method was linear over concentration range 0.1 to 3 µg/ml, 0.5 to 6 µg/mL, 0.2 to 2.5 µg/mL and 0.1 to 1.5 µg/mL of gemifloxacin mesylate, theophylline, warfarin and omeprazole, respectively. The mobile phase was selected as mixture of dichloromethane, methanol and ammonia in the ratio of (7: 5.5: 3 v/v/v) for the development of plates. Densitometric analysis was carried out at wavelength 254 nm. The stability of gemifloxacin mesylate and the co-administered drugs in plasma was confirmed during three freeze-thaw cycles (−20°C).