Ofloxacin is an antibiotic from second generation of fluoroquinolone's group. Concentration of ofloxacin in human plasma is low, so it requires a selective, accurate, sensitive method for analysis it. In this study, the optimization and validation of ofloxacin analysis in human plasma using high performance liquid chromatography-fluorescence with ciprofloxacin-HCl as an internal standard were carried out. Separation of ofloxacin was performed using C18 (Waters, Sunfire TM 5 µm; 250 x 4.6 mm) column with an isocratic mobile phase consisted of triethylamine 1% on water pH 3.0–acetonitrile (84:16) an in the flow rate of 1.0 mL/min, and on column temperature 40oC whereas the detection was carried out at excitation of 300 nm and emission of 500 nm. Plasma extraction was done by deproteination using methanol, through the process of vortex and centrifugation (10000 rpm) for 2 minutes and 10 minutes consecutively. The method was valid and linear within the concentration ranged from 21,4 ng/mL to 4280 ng/mL with LLOQ of 21,4 ng/mL. Intra-day and inter-day accuracy and presicion was not more than + 20% for LLOQ and not more than + 15% for QCL, QCM, and QCH samples in both % diff and coefficient of variation. Ofloxacin was stable in human plasma at least three freeze and thaw cycle, for at least 24 hours in room temperature and 28 days at -20oC. This bioanalytical method fulfilled the acceptance criteria following EMEA guideline.