The multivariate calibration method partial least squares regression type 1 (PLS-1) was applied to the simultaneous fluorimetric determination of pyridoxal, pyridoxamine and pyridoxic acid at low concentration levels (8.0–80, 30.0–300 and 2.4–24 µg l^–1, respectively). The non-linear variable-angle synchronous spectra were obtained, tracing a curved trajectory through the total luminescence spectroscopic data. Several calibration models were studied in order to select the best one. The statistical parameters obtained by internal and external validation of the optimum calibration model were in agreement, with satisfactory results.