A reverse phase HPLC procedure is described for the determination of chloramphenicol and its hydrolytic product, 2-amino-1-(4-nitrophenyl)-propane-1,3-diol. The method was validated for reproducibility, linearity and recovery for both components. It was successfully applied to monitor post-marketing stability of ten batches of chloramphenicol ophthalmic solutions stored at room temperature. As compared to the BP method using solvent extraction and UV measurements, for the determination of chloramphenicol and its hydrolytic product in chloramphenicol eye drops, the suggested procedure was found to be relatively more accurate, precise and less time-consuming.