Heparin and heparan sulfate (HS) are linear polyanions from the glycosaminoglycan (GAG) family. They are conceived to play critical roles in a variety of biophysiological processes by interacting with key proteins and regulating protein functions. Understanding their biological functions and exploit heparin’s unique versatility for therapeutic purposes critically depend on the characterization of their interactions with relevant proteins, which, however, is largely impeded by the structural heterogeneity of these polyanions. This work presented the development of novel mass spectrometry-based analytical strategies incorporated with liquid chromatography and ion mobility spectroscopy to investigate heparin-protein interactions and address their significance from different aspects.