Cholesterol (CLR) has been shown to modulate membrane dynamics and alter integral membrane protein (IMP) function. However, understanding the molecular mechanisms of these processes is complicated by limited and conflicting structural data: Specifically, in co-crystal structures of CLR-IMP complexes it is difficult to distinguish a specific CLR-IMP interaction with biological relevance from a nonspecific association visible under the non-natural circumstances of the crystallization conditions. We leverage the increasing number of experimental CLR-IMP structures in the Protein Data Bank to systematically analyze putative interaction sites based on their tertiary structure and evolutionary conservation. This information is implemented into RosettaLigand to underpin future development towards detecting and engineering CLR-IMP interaction sites.