Drug-induced phospholipidosis (PLD) is a lipid storage disorder characterized by reversible accumulation of phospholipids in tissues. It is generally considered to be an adaptive response upon administration of certain drug-typically cationic amphiphilic drugs (CADs)-rather than a toxic manifestation. However, tissue and organ damage have been reported in association with PLD for some cases. There is only a limited pool of available screening methods to predict a drug’s PLD-inducing potential. We have developed a high-throughput fluorescence non-cell based assay to screen for the drug-phospholipid interaction, which correlates to phospholipidosis. Anionic amphiphilic phospholipids can form complexes in aqueous solution and its critical micelle concentration (CMC) is determined using the fluorescence probe N,N-dimethyl-6-propionyl-2-naphthylamine (Prodan). Upon interaction with drug candidates, this CMC may shift to a lower value due to the association between lipids and drug candidates. The relative change, CMCDL/CMCL, provides a direct measurement of the drug-lipid association. Metabolism of a drug can change the degree of phospholipidosis depending on the rate of metabolism and the nature of the metabolite(s). Our data from forty-five drugs, along with metabolites of ten drugs, in this fluorescence assay demonstrate a good correlation with phospholipidosis as reported in cellular assays, in vivo tests, and human studies in the literature. We therefore report the first fluorescence based non-cell in vitro assay to screen for PLD-inducing potential in drug candidates, which is additionally fast, reliable and cost-effective.