Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal COFRADIC technology and identified 46 shared, 3 caspase-3-specific and 6 caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40S ribosomal protein S18, was studied in detail. The Rps18-derived P6-P5’ undecapeptide retained complete specificity for caspase-7. The corresponding P6 P1 hexapeptide still displayed caspase-7 preference, but lost strict specificity, suggesting that P’ residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in case of Rps18 the P4-P1 residues constitute the core cleavage site but that P6, P5, P2’ and P3’ residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 essentially relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.