An HPLC method for the analysis of nitrofurantoin (NTR) in human urine has been developed and evaluated. This method is based on a selective extraction and reverse-phase chromatography which results in chromatograms with 2400 to 2500 theoretical plates for NTR and the internal standard (Furazolidone). The method is highly reproducible and has been validated over the range of 2 to 400 mcg/ml. A significant factor in the assay of nitrofurantoin, not common to most drugs, is that urine concentrations can exceed the aqueous solubility of the compound. Thus, special consideration in the handling of samples is needed to assure accuracy and reproducibility in the analysis. This method requires the pipetting of samples into precise aliquots prior to freezing and storage.