The aim of this paper is to develop and validate an HPLC method to investigate the tissue distribution of gemifloxacin (GEM) in rats following intravenous administration and compare the penetration of this drug in different tissues undergoing normobaric and hyperbaric oxygen (HBO) exposure. The chromatographic conditions consisted of a C 18 column and mobile phase composition of triethylamine (0.5% v/v, pH 3.0), methanol and acetonitrile (65:28:7, v/v/v) at a flow rate of 1.0 mL min −1 . The calibration curves were linear from 0.2 to 30 μg mL −1 for GEM, with correlation coefficient r  ≥ 0.99. Retention times of GEM and furosemide (internal standard) were approximately 8 and 16 min, respectively. The intra-day variation was less than 5.56, 8.94 and 5.74% for lung, kidney and liver, respectively, and the inter-day variation was less than 1.58, 3.71 and 3.45% for lung, kidney and liver, respectively. The accuracy ranged from 89.0 to 112.9%. Recoveries of GEM and furosemide were up to 90%. Short-term, freeze–thaw, long-term and autosampler stability were demonstrated. The present study showed that the highest tissue concentration of GEM under normobaric exposure was obtained in the kidney (51.22 μg g −1 ), followed by liver (32.78 μg g −1 ) and lung (28.49 μg g −1 ). The t 1/2 β obtained was 6.35, 2.83 and 2.41 h for lung, kidney and liver, respectively. Statistical difference ( p  < 0.05) was observed in rat tissues—lung, liver and kidney, when the rats were exposed to normobaric and HBO (2.5 h at 1.7 ATA).