Calpains are intracellular proteases that selectively cleave proteins in response to calcium signals. Although calpains cut many different sequences, residue preferences within peptide substrates were recently determined and incorporated into a superior FRET (fluorescence resonance energy transfer)-based substrate (PLFAER). Here we show PLFAER is cleaved by calpain at the intended F-A scissile bond. Sequential replacement of individual residues by alanine reduced activity except with PLFAAR, which is cleaved 2.3 times faster than PLFAER. The rates of hydrolysis of the alanine-substituted substrates were used to compare substrate preferences of calpain, papain and cathepsins B and L. The preferences of the two major isoforms, calpains 1 and 2, were virtually indistinguishable and were very similar to those of the calpain 1 protease core and papain. However, the activity profiles with the FRET substrate series were significantly different for the cathepsins, particularly cathepsin B.