Since the analysis of urinary neopterine is important in diagnosing malignancy, a method has been developed for its rapid and sensitive separation and quantitation using high-performance liquid chromatography (HPLC) on reversed phase. Eluted neopterine is monitored by fluorescence at a excitation wavelength of 353 nm and measured at 438 nm. Separation was optimized by elution with 15 mmol/l potassium phosphate (pH 6.4) at a flow-rate of 0.8 ml/min. Urinary neopterine was related to creatinine with the aim of reducing variations due to fluctuating urinary concentrations. The proposed method has good performance characteristics and is not influenced by the presence of reduced neopterine in urine. Using this HPLC method, urinary neopterine related to creatinine was determined for 148 healthy male adults (mean neopterine/creatinine 113 mumol/mol), 146 healthy female adults (mean neopterine/creatinine 140 mumol/mol) 60 healthy children (mean neopterine/creatinine 163 mumol/mol). The neopterine levels for four healthy individuals were measured daily over a period of one month.