Abstract: A method of liquid chromatography-mass spectrometry was developed for separation and differentiation of the Z/E isomers of bulk cefprozil and its intermediate substance (6R,7R)-7-amino-8-oxo-3-(1-propenyl)-5-thia-1-azabicyclo4.2.0oct-2-ene-2-carboxylic acid (7-APRA). For the 7-APRA isomers, both of the deprotonated molecules are favorable to undergo fragmentation via losing H₂S and OCNH, and give the product ions at the m/z 161 and m/z 152, respectively. Different to the E isomer, the Z isomer of 7-APRA is facile to undergo γ-H migration, which promotes the H₂S elimination. As a result, Z-7-APRA has the significantly higher abundance ratio of I161/I152 than the E isomer, which can be used for isomer differentiation. Analogously, the deprotonated cefprozil Z isomer is also more favorable to undergo dissociation to form the ion of m/z 205, due to the phenol function group in the structure. Furthermore, their fragmentation mechanisms were proposed, and the density functional theory (DFT) calculations were employed to consist with the experimental results.