The aim of this study was to develop an algorithm suitable for the estimation of 4-pyridoxic acid (4PA) concentration removed during dialysis through fluorescence measurements in spent dialysate. 4PA is the major urinary catabolite of vitamin B-6 (B6), that is regularly injected to dialysis patients. 4PA could be used to estimate B6 nutritional status of the patients. For the study 10 hemodialysis patients during 40 dialysis sessions were studied. Full fluorescence spectra of spent dialysates were recorded with the spectrofluorophotometer RF-5301 by Shimadzu (Kyoto, Japan) and 4-pyridoxic acid concentrations were measured with HPLC system. Two single wavelength models were developed were correlation coefficients for Ex/Em 320/430 nm were 0.931 and 0.853 and for Ex/Em 310/460 nm were 0.944 and 0.931 for calibration and validation set, respectively. In summary, 4PA concentration can be estimated through fluorescence measurements from spent dialysate.