The identification of new antifungal molecules is an important goal of current anti-infective research. To achieve this goal, alternatives to traditional growth inhibition-based screening have been developed in recent years. In this study, we describe an assay to detect molecules that disrupt yeast cell integrity by using the release of adenylate kinase (AK) into culture medium as a reporter of yeast cell lysis. The protocol is applicable to 96- and 384-well microtiter plate formats; uses a commercially available luminescence assay kit to detect AK activity; is more sensitive than traditional growth-based assays; and is specific for fungicidal compounds. In the high-throughput setting, the procedure provides excellent Z' scores (0.75-0.9), making it a highly robust assay. The AK assay is performed in a single microtiter plate using an 'add and read' procedure that can be completed in a single work day.