Parkinson’s disease is the second most common neurodegenerative disorder worldwide and, despite daunting efforts, remains incurable. α-Synuclein (α-Syn) aggregation into intracellular inclusions is the main pathological hallmark of this disease and, accordingly, is an important therapeutic target. Due to the disordered nature of the protein, high-throughput screening has become the gold-standard approach for identifying α-Syn aggregation inhibitors. Here, we describe a comprehensive protocol for developing α-Syn aggregation modulators. This approach combines a straightforward and highly reproducible method for screening large chemical libraries with a suite of orthogonal validation techniques, cytotoxicity assays, and in vivo testing. This strategy, which has crystallized in the discovery of potent α-Syn aggregation inhibitors, can be readily adapted by other laboratories, thus contributing to finding disease-modifying drugs for Parkinson’s disease.