Current methods typically used for metabolite screening and disease diagnosis often require extensive sample preparation, which increases analysis time and associated costs. While ambient ionization techniques enable the analysis of various samples in complex matrices with little or no sample preparation in a short time (typically within a minute), their reduced selectivity, even when coupled with high-resolution mass spectrometers, limits their application in certain fields. In this study, we have optimized the coupling of paper spray (PS) and leaf spray (LS) ambient ionization techniques with a commercially available ion mobility mass spectrometer (IM-MS) and demonstrated the separation of geometric and constitutional isomers. Ambient ionization techniques allow simultaneous introduction and ionization of samples, while background noise and matrix interference from paper and leaf substrates are filtered out by IM separation, resulting in high sensitivity and selectivity of the PS-IM-MS and LS-IM-MS workflows. In addition, we introduced a novel approach to perform single-field collision cross section (CCS) measurements, which resulted in CCS values that differ by 0.15% and 0.25% from traditional stepped-field and single-field methods, respectively. In addition, we used advanced computational tools to confidently identify analyte structures by comparing CCS values from experimental IM measurements and theoretical calculations. These results suggest that the coupling of ambient ionization methods with ion mobility techniques enables rapid, sensitive, and highly selective analysis that can be used in different fields, such as agrochemical screening and disease diagnostics.