Abstract: The related substances in folic acid were separated on an ODS column by linear gradient elution using the mixture of 10 mmol/L ammonium acetate buffer solution and methanol (pH 6.3) as the mobile phase, and their accurate mass-charge ratio and elemental composition of parent and product ions were detected by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF/MS). The structures of the related substances were then determined by MS spectra elucidation, organic reaction mechanism analysis, or reference substances confirmation. Under the established LC-ESI-Q-TOF/MS conditions, folic acid and its related substances were adequately separated. Among the 23 main related substances found, 2 were known impurities listed in European Pharmacopoeia, 4 were consistent with the literature reports, and the remaining 17 were newly identified unknown related substances in this study. The results of separation and identification can provide some useful reference for the quality control of folic acid.