Abstract: A simple, sensitive isocratic method for detection and quantification of ofloxacin in plasma has been developed. The assay consisted of reversed HPLC with UV detection. Separation was achieved on a C18 column. The detection limit was 10 ng mL–1 in plasma. The validation data has been studied. This method provides sufficient sensitivity for pharmacokinetic studies and dose control in veterinary as well as in human medicine.