To investigate the validity of using HPLC procedure with the fluorescence detection for the estimation of the pyridoxal 5'-phosphate (PLP) concentration in human blood plasma, we modified the conditions of our previous HPLC method for PLP. By changing the pH of the mobile phase (pH 5.5) and by altering the detection wavelengths in fluorescence (excitation at 322nm and emission at 417nm), both the improved sensitivity for pyridoxic acid 5'-phosphate and elimination of interfering extraneous peaks were attained. We reported the total vitamin B₆ content of healthy adult volunteers obtained by this HPLC method.