Ion mobility separations are based on fundamental principles that differ from liquid chromatography (LC) in that several parameters of ionized molecules—size, charge, shape, and structure—come into play simultaneously. As a result, analytes that have the same molecular mass can be separated by their shape, charge and collision cross section (CCS). A new development in ion mobility separation, high-resolution ion mobility (HRIM), overcomes the problem of ion loss in other ion mobility separation techniques. HRIM also provides excellent structural resolution and high reproducibility. The technique is particularly well suited to challenging applications in the pharmaceutical and clinical fields, such as glycosylation monitoring of biological drugs and vitamin D analysis.