Cephalosporins remain one of the largest classes of antibiotics used clinically, with a diverse group of dual substituents at C3 and C7 positions. The fragmentation of the C-3 side chain from the C3' position after hydrolysis by Serine or metallo-beta-lactamase, leaving an exo-methylene group, has been introduced in the literature recently, but the exact mechanism by which it happens remains unclear until now. Our incrystallo findings suggest that the fragmentation of the C3 side chain will depend on the C3' atom and its hybridization state. A C3' carbon with sp2 hybridization will not allow fragmentation, but a sp3 hybridized carbon will facilitate the detachment of the side chain. Furthermore, heteroatoms like Sulfur, on C3' position, bearing vacant d-orbitals participate in pi cloud delocalization, making the detachment of C3 side chain energetically unfeasible. For the sp3 hybridized carbon at C3', the fragmentation of the bond was independent of the C3'' atom. We observed fragmentation of the C3'-C3'' bond for all three heteroatoms, Nitrogen, Oxygen, and sulfur, at the C3'' position. We validated our crystallographic findings with mass spectrometry to confirm the fragmentation of the C3 side chain in sp3 carbon and the unfragmented side chain in sp2 carbon and sulfur at C3' position. QM/MM simulations on three types of enzyme-cephalosporin complexes with different C3 side chains confirm the mechanism of fragmentation. Beyond revealing C3 side chain fragmentation in cephalosporins, useful for inhibitor and diagnostic design, our QM/MM methods are useful on incrystallo complexes to show the en-zyme catalysis.