A strategy for the screening and optimization of an artificial esterase is presented that utilizes the self‐assembly of amphiphilic molecules. Unlike conventional approaches that rely on the attachment of key functional groups onto molecular scaffolds or surfaces, the modular assembly of amphiphiles allows a large number of catalytic combinations to be investigated with minimal synthetic effort. In this study, iterative combinatorial screens led to an optimized esterase comprising amphiphiles that act as a nucleophilic catalyst, an oxyanion hole and a metal ion chelator. Cooperativity is observed between the functional headgroups of the amphiphiles, an effect that is diminished when co‐assembled with non‐functionalized surfactants. Assessment of the catalytic efficiency (kcat/KM) of our optimized catalysts against recently reported artificial esterases shows comparable efficiency, indicating that efficient catalysis is possible with dynamic self‐assembled systems despite the absence of pre‐defined rigid binding pockets.