Cholesterol is an integral component of cellular membranes and its precise intracellular concentration and distribution is essential for maintaining cellular function. Cholesterol trafficking between cellular membranes, for example from its site of synthesis in the endoplasmic reticulum (ER) to the plasma membrane (PM) where it represents one out of every 2-3 lipids, occurs mainly by non-vesicular transport and requires carrier proteins. Known sterol transport proteins, including the oxysterol binding protein and its homologs, and StARkin proteins with sterol-binding StART domains are not physiologically essential in transporting cholesterol between the ER and PM, the route of biosynthetic sterol transport. Thus, the full complement of cytosolic sterol transport proteins (STPs) remains to be identified. Hypothesizing that STPs must first and foremost be sterol binding proteins (SBPs), we used in vivo labeling to demonstrate the presence of SBPs in yeast cytosol. Next, via an in vitro chemo-proteomic strategy involving a photo-reactive clickable cholesterol analog and quantitative mass spectrometry, we identified 342 putative SBPs. In-depth analysis of the identified proteins based on their annotated function, reported drug phenotypes (amphotericin sensitivity), reported interactions with proteins regulating lipid metabolism, gene ontology enrichment (sterol/lipid/signaling function), and conserved mammalian orthologs revealed a subset of 50 characterized and 9 uncharacterized proteins. The uncharacterized proteins are found to be involved in maintaining plasma membrane integrity based on their growth in the presence of nystatin and myriocin, drugs that target sterols and sphingolipids, respectively. The dataset reported here constitutes a comprehensive resource for functional analysis of sterol binding/transport proteins and may provide insights into novel aspects of non-vesicular sterol trafficking.