A simple, fast and precise method for the simultaneous determination of neopterine (Npt), pseudouridine (Psu) and creatinine (Cre) in urine by using ion-pair HPLC has been developed. The urine specimen is subjected to a microcolumn clean-up step, providing a ten-fold sample dilution, and is then injected into the column. The eluate is forced through the respective cells of a fluorescence and then a UV detector, which are connected in series. Neopterine is monitored by fluorescence emission at 438 nm (excitation wavelength 353 nm) while Psu and Cre are monitored by UV absorption at 235 nm. This allows the determination of Npt/Cre and Psu/Cre concentration ratios in a single run on the same sample, and the use of urine specimens collected randomly instead of 24 hourly collections. The method has been applied to urine specimens from 19 healthy, male donors (mean Npt/Cre (mumol/mol) and Psu/Cre (mumol/mmol) values of 41.7 and 16.8, respectively) and 23 healthy, female donors (mean Npt/Cre (mumol/mol) and Psu/Cre (mumol/mol) values of 67.4 and 18.5, respectively). The within-run S(r) for Npt/Cre and Psu/Cre concentration ratios ranged between 3.3 and 4.6%.