ABSTRACTPeptide‐based nanomolecular constructs offer great possibilities for designing catalytic molecular systems mimicking enzymes. In this study, we designed three tripeptide catalysts that can possibly mimic hydrolase enzymes, with the objective of systematically verifying the scope of modulating enzymatic activity. Histidine residue was placed at three different locations in Fmoc‐tripeptide sequences, thus generating three chemically similar but sequentially different molecules, P1, P2, and P3. From our study, the peptide catalyst P3 has shown maximum catalytic activity with a chromogenic substrate, p‐nitrophenyl acetate, that gets hydrolyzed to p‐nitrophenol. The catalytic activity has increased with an increase in pH and temperature, though pH dependency cannot be generalized and can vary depending on the reaction mechanism. Importantly, this study successfully demonstrates the possibility of modulating the activity of functional mimics of bioactive molecules by tuning the principal components of functional molecules.