The separation of small molecule isomers has become a proving ground for ion mobil-ity spectrometry (IMS). To date, successful IMS separation of steroid isomers hasbeen accomplished largely by examination of alkali metal cationizeddimers, with fewexamples of successful separations ofmonomericsteroid isomers in either positive ornegative ion modes. Here, we report on the novel use of anion attachment to formnegative ion monomeric adducts of steroid isomers that may be separated by IMS.Computational modeling shows that [prednisolone + Cl]−adopts a conformationwherein the attaching chloride is chelated between two hydroxyl hydrogens. Theelectrostatic interaction between Cl−and the two substituent electropositivehydroxyl hydrogens causes“conformation pinning”of prednisolone, thereby forcingthe complex to adopt a constricted conformation. The tighter conformation of [pred-nisolone + Cl]−permits baseline IMS separation from its isomer [cortisone + Cl]−thatexists only in elongated form. Although distinguishable as anionic adducts, these iso-meric steroids were impossible to separate as either protonated (MH+, positive mode)or deprotonated ([M-H]−, negative mode) analogs. Another pair of isomeric steroids21-deoxycortisol and corticosterone showed improved IMS separations as chlorideadducts as compared to the analogous MH+or [M-H]−pairs. Lastly, success wasshown in separatingprotonateddimeric forms of isomeric steroid pairs by IMS, andwe distinguish these separations from those of alkali metal cationized dimers thathave been previously reported.