A validated, simple and universal HPLC-UV method for the determination of cimetidine, famotidine, nizatidine and ranitidine in human urine is presented. This is the first single HPLC method reported for the analysis of all four H 2 antagonists in human biological samples. This method was also utilized for the analysis of ranitidine and its metabolites in human urine. All calibration curves showed good linear regression (r 2 >0.9960) within test ranges. The method showed good precision and accuracy with overall intra- and inter-day variations of 0.2–13.6% and 0.2–12.1%, respectively. Separation of ranitidine and its metabolites using this assay provided significantly improved resolution, precision and accuracy compared to previously reported methods. The assay was successfully applied to a human volunteer study using ranitidine as the model compound.