3D printed PLA has already been demonstrated for several biotechnological applications, including enzymes immobilization. The prerequisites for an efficient screening assay include using small volumes of reagents, low cost, and rapid screening of large numbers of compounds and extracts. Hence, assays based on microtiter plates are predominant. Thus, designing and fabricating scaffolds on a similar scale, which could serve as immobilization carriers, and their recruitment in inhibitors screening studies is of great significance, adding both enzyme stability and reuse potentiality of the biocatalytic system in assay merits. In this work, pancreatic lipase was immobilized on 3D-printed PLA microwells for enzyme inhibitor screening. XPS analysis demonstrated the successful modification of the PLA scaffolds. The immobilized enzyme displayed high levels of operational, thermal, and storage stability under the tested conditions. The IC₅₀ values for PPL inhibition were calculated for Orlistat, a model lipase inhibitor, and olive leaf extract, a promising natural compound. This is the first study reporting the use of 3D-printed PLA wells with an immobilized enzyme for inhibitor screening assay.