Lysosomes are a type of functional compartment, or organelle, that is responsible for crucial cellular processes such as protein homeostasis, nutrient sensing, molecular signaling, and secretion. These biochemical pathways ultimately dictate the intricate balance between health and diseases, including neurodegeneration and cancer. Despite decades of research on lysosomes, their role in devastating disorders such as Alzheimer’s disease remains largely unknown. In order to achieve a better understanding of lysosomes, specifically in the context of therapeutics for neurodegeneration, my thesis aimed to (1) describe the exciting field of organelle probe development for phenotypic discovery campaigns, (2) engineer a novel biochemical probe that measures lysosomal pH, a crucial aspect of lysosomal function in cells and (3) launch a high-throughput drug screen focused on identifying small molecules and possible molecular pathways that govern lysosomal function. Ultimately, this work contributes to the actively expanding field of phenotypic drug discovery, with specific focus on lysosomal pH and development of cell-based quantitative techniques.