A simple and isocratic HPLC method with stability indicating nature was developed and then subsequently validated for simultaneous determination of ciprofloxacin and dexamethasone in pharmaceutical formulations, human serum and urine. Best chromatographic separations were attained within run time of 10 minutes using C8 as stationary phase and mixture of phosphate buffer and methanol (41:59 v/v) as mobile phase. The mobile phase was flowed at 1.5 mL min-1 with detection of both the analytes at 270 nm using photodiode array detector. Validation of the method was accomplished using specificity, linearity, accuracy, precision, robustness, LOD and LOQ. The method was found linear from 3-21 mg mL-1 for ciprofloxacin (r2 ≥ 0.999) and 1-7 mg mL-1 for dexamethasone (r2 ≥ 0.999). The %age recoveries of ciprofloxacin in spiked human urine and serum were ≥99% and ≥85% respectively, while for dexamethasone they were ≥97% in both matrices. The method proficiently separated the peaks of ciprofloxacin and dexamethasone from all types of interfering substances including degradation products/impurities with purity index ≥ 0.9998. The method thus was stability-indicating and can be employed for simultaneous analysis of ciprofloxacin and dexamethasone in complex matrices involving multiple components in the mixture.