Updated project metadata. Integral membrane proteins (IMPs) are permanently embedded within the plasma membrane and execute multiple cellular functions. IMPs constitute important targets, but development of drugs that target IMPs to modulate protein–protein interactions, which mediate their functions, has been challenging. Characterizing the structural and functional properties of IMPs as well as their regulatory PPIs is a key step toward new or improved therapeutics. Functional and structural studies of IMPs are hindered by their hydrophobicity, low expression levels in cells, flexibility, and the need to use detergents to solubilize these proteins. Representative examples of IMPs are mammalian adenylyl cyclases that play a pivotal role in the 3',5'-cyclic adenosine monophosphate signaling pathway. The activity of membrane-bound adenylyl cyclases can be regulated by direct and/or indirect binding of calcium ions, for example, via calmodulin. Unstructured and highly flexible regions of adenylyl cyclases engage in PPIs. However, the principles of classical proteomics methods do not allow mapping interactions involving regions without a defined three-dimensional structure. To address this limitation, we used limited proteolysis–mass spectrometry (LiP–MS) to study the known interaction between the adenylyl cyclases AC8 and calmodulin by probing protein structural alterations in crude membrane suspensions treated with calmodulin. The LiP–MS analysis pinpointed putative binding site regions of AC8 previously shown to participate in the interaction with calmodulin, demonstrating that this method can be used to identify interaction domains in membrane proteins in a physiologically relevant context.