A simple bioanalytical reversed phase HPLC method for the determination of the ciprofloxacin in serum, aqueous humour and ophthalmic drops was developed and validated. HPLC equipped with Hypersil C18 Column was operated in isocratic mode using acetonitrile and 0.25 M H3PO4 (60:40 v/v) as mobile phase that was pumped at rate of 1ml.min-1 and eluents were monitored using UV-Visible detector at 275 nm. The Ciprofloxacin was analysed in serum, aqueous humour and ophthalmic drops using acetaminophen as an internal standard (IS). The instrumental response was linear in the concentration range of 5 ng.ml-1 to 75 ng.ml-1 in aqueous humour and 10 ng.ml-1 to 70 ng.ml-1 in serum. The LOD and LOQ, precision and reproducibility of the method were good for pharmacokinetics studies.