Neurodegenerative disease is characterized by the presence of various aggregated and misfolded protein species. Alpha-synuclein (αS) aggregation is a prominent example, with implications in disorders such as Parkinson’s disease (PD), multiple system atrophy (MSA), and Lewy body dementia (LBD). Despite advancements in small molecule targeting of these proteins, the binding-site landscape remains poorly understood due to aggregate polymorphism and cryptic binding pockets. To this end, photoaffinity labeling (PAL) is a method aimed at determining a small molecule’s protein target or binding site by covalently trapping the binding interaction under UV irradiation. This method was implemented using αS pre-formed fibrils (PFFs) in conjunction with a variety of photo-crosslinker derivatives of small molecule ligands. Binding sites were determined using liquid chromatography paired with tandem mass spectrometry (LC-MS/MS), which enabled further rationally designed structure activity relationship (SAR) studies towards positron emission tomography (PET) diagnostic probes for PD and for MSA. To bolster future PAL studies, a novel photo-crosslinking motif was identified and developed. Isoxazoles were shown as robust photoaffinity probes using PFFs as a model system, and additional chemoproteomic studies were conducted using a photoaffinity variant of the isoxazole-containing FDA-approved antibiotic sulfisoxazole. Proximity labeling (PL) is a method for determining a protein’s interaction partners (interactome) in a cellular context. This technique uses a functional attachment to a protein of interest which enables covalent tagging and subsequent enrichment of interacting proteins. To achieve this with αS, the µMap PL system was adopted, which involves attachment of an iridium photo-catalyst (Ir) to an antibody or protein of interest that serves as an antenna for blue-light sensitization of a nearby diazirine probe. This probe can then form a carbene, which covalently inserts into nearby proteins with a uniquely small labeling radius. Several diazirine probe molecules were synthesized for fluorescent labeling and streptavidin enrichment. These were benchmarked using αS functionalized with Ir at multiple positions, and a hybrid experimental/computational carbene labeling radius was determined using PyRosetta paired with LC-MS/MS crosslinking data. Finally, the site-dependent interactome of αS in a total brain homogenate was investigated using a quantitative bottom-up proteomic approach.