Cephalosporins are a widely used subclass of β-lactam antibiotics that demonstrate variable protein binding independent of generation or antibiotic coverage. Prior work analyzed carbon 3 (C3) and carbon 7 (C7) substituents (locations of R2 and R1 groups respectively) for protein binding interactions. This study builds upon these results with statistical analysis of additional agents of the class. Chemical structures of 23 cephalosporins were used to identify the presence of 40 functional groups, and correlative relationships were identified using established protein binding data. Four functional groups were significantly correlated with protein binding: tetrazole (positive association), pyridinium, primary amine, and quaternary amine (negative associations). Cephalosporins with a negative charge at physiological pH were associated with increased protein binding. Analysis of tetrazole-containing cephalosporins and ceftriaxone indicates the need for further study of the potential role in protein binding of neutral or negatively charged aromatic nitrogen heterocycles linked at the C3 position by a thiomethylene group.