Disorder in proteins, or in extended protein regions, is more commonplace and functionally relevant than was conventionally assumed. Until a few decades ago, well-defined structure was considered as the only prerequisite for properly functional proteins. Since the development of disorder predictors, it has become apparent that intrinsically disordered proteins (IDPs) comprise significant proportions of eukaryotic genomes. An increasing number of investigations into the structure and function of IDPs reveal that they mediate a large number of crucial cellular processes and are associated with various disease processes. Despite the growing interest in IDPs, much remains unclear about the relationships between IDP structures, their interactions with other proteins and their mechanisms of action. Another important consideration is whether IDPs remain disordered in crowded conditions, such as the native cellular environments where IDPs function. Mass spectrometry (MS)-based approaches have been developed to probe IDP secondary structure and to complement other biophysical techniques in elucidating IDP functions. In addition, liquid-chromatography (LC) provides avenues for the removal of interference from complex protein samples containing high concentrations of polymeric crowding agents. Described herein, are studies based on hydrogen exchange (HX) measured by mass spectrometry (HX-MS) to: (1) propose a mechanism for calcineurin activation based on structural changes in its disordered regulatory domain, occurring upon binding calmodulin, (2) develop a method to probe the effects of polymer crowders on IDPs, and (3) investigate the effects of different crowder concentrations on transiently helical regions of random coil IDPs.