This study presents an analytical approach coupling novel ambient ionization sources with trapped ion mobility spectrometry (TIMS) and tandem mass spectrometry (MS/MS) for the rapid characterization of fentanyl analogs. Two ambient ionization sources were illustrated for minimal sample preparation and rapid analysis: electrospray ionization (nESI) and direct analysis in real time (DART). Fentanyl analogs can be separated using nESI-TIMS-MS/MS based on differences in their mobility and/or fragmentation pattern; reference mobility spectra are reported for 234 single standards. In contrast, DART-TIMS-MS/MS allowed for the characterization of 201 compounds due to differences in the protonation pattern and efficiency when compared to nESI. The TIMS high resolving power (R > 80) allowed baseline separation for most isomers and mobility trends were established for methylated and fluorinated isomers, with the more compact ortho-substituted analogs showing distinct separation from para- and meta-substituted species. This multi-dimensional strategy offers a comprehensive characterization of fentanyl analogs and other synthetic opioids with minimal sample preparation. This analysis shows significant potential for high-throughput screening (<5 min) and high sensitivity detection (