The substrate specificity of CPB2.8ΔCTE, a recombinant cysteine protease from Leishmania mexicana, was mapped by screening a fluorescence‐quenched combinatorial peptide library. Results from library screening indicated a preference for Arg or Lys in the S3 subsite and for hydrophobic residues, both aliphatic and aromatic, in S2. The S1 subsite exhibited a specificity for the basic residues Arg and Lys. Generally, the specificity of the primed subsites was less strict compared with the non‐primed side which showed preference for Arg, Lys and Ala in S$_{1}^{\prime }$, Arg, Pro and Gly in S$_{2}^{\prime }$ and Lys, Arg and Ser in S$_{4}^{\prime }$. By contrast, a strict preference for the basic residues Arg and Lys was found for S$_{3}^{\prime }$. Overall, there was a trend for basic residues in alternating subsites and smaller residues in the primed sites compared with the non‐primed sites. In addition, there were strict requirements for the amino acids in subsites S3–S1. Fluorescence‐quenched peptides from the library with the highest on‐resin cleavage were resynthesised and their kinetics of hydrolysis by CPB2.8ΔCTE assessed in solution phase assays. Several good substrates containing the quintessential dipeptide particular to cathepsin‐L‐like enzymes, ‐F‐R/K‐, in P2 and P1 were identified (e. g. Y(NO2)‐EKFR↓RGK‐K(Abz)G, Abz=2‐aminobenzoyl; kcat Km−1=4298 mM−1 s‐1). However, novel substrates containing the dipeptide ‐L/I‐Q‐ in P2 and P1 were also well hydrolysed (e. g. Y(NO2)‐YLQ↓GIQK‐K(Abz)G; kcat Km−1=2583 mM−1 s‐1). The effect of utilising different fluorescent donor–quencher pairs on the value of kcat Km−1 was examined. Generally, the use of the Abz/Q‐EDDnp donor–quencher pair (EDDnp=N‐(2,4‐dinitrophenyl)ethylenediamine) instead of K(Abz)/Y(NO2) resulted in higher kcat Km−1 values for analogous substrates.