The methodology for obtaining accurate ion-neutral collision cross section (Ω) values for peptides and proteins using periodic focusing ion mobility spectrometry (PF IMS) is presented. A mobility dampening factor (represented by the term α) is introduced to account for the relative increase in ion-neutral collisions in PF IMS compared to uniform field ion mobility spectrometry (UF IMS) for equivalent operating conditions. The results show that α may be easily quantified both theoretically and empirically for a specific PF IMS design operating at a given pressure based upon the charge state of the analyte. By simply incorporating an α term into traditional UF IMS expressions, accurate Ω values were obtained with excellent agreement (≤4% difference) compared to UF IMS measurements found in the current literature.