Since no proper method is available in literature for the analysis of pyridoxal-5'-phosphate, a reversed phase liquid chromatographic method was developed and validated for specificity, sensitivity, linearity, precision and accuracy. Nine potential related substances and forced degradation products could be successfully separated from the main peak. The separation was achieved on a Polaris C18 column (250 × 4.6 mm i.d., 5 μm) using a mobile phase consisting of 20 mM ammonium formate in 0.65% formic acid - acetonitrile (98.8:1.2, v/v). Isocratic elution was performed at a flow rate of 1.0 mL/min and the analytes were detected by UV at 240 nm. The volatile mobile phase allowed also direct coupling to an ion-trap mass spectrometer with a positive electrospray ionization source to characterize unknown peaks in the chromatogram. The method can be used for quality control purposes as required by regulatory authorities to ensure the product's safety and efficacy.