Mass spectrometry has emerged as a key technology for both untargeted and targeted analysis of molecules in nature, including metabolites, lipids, proteins, pollutants, and synthetic chemicals. However, conventional tandem mass spectrometry with collision-induced dissociation (CID) is challenged by the unambiguous identification of isomers. To overcome this challenge, radical-driven fragmentation techniques, such as ultraviolet photodissociation (UVPD) and electron activated dissociation (EAD), have become essential tools for the structural elucidation of biomolecules, revealing structural features, such as double bond positions, that are inaccessible through CID of even-electron precursor ions. While the fragmentation of odd-electron radical cation precursors, often observed in EAD and UVPD, has been shown to access orthogonal structural features, little effort has been made to enhance radical cation formation within the ion source for subsequent MS2 experiments. Atmospheric pressure photoionization (APPI) is well suited for the generation of radical cations through charge-exchange reactions. To maintain radical cations, it is necessary to limit proton-transfer reactions with and ionization of solvent constituents in the gas phase. APPI avoids ionization of typical LC solvents and is less affected by undesired gas-phase reactions than other ionization techniques. To further reduce undesired gas-phase reactions and enable adaptive ion formation, it is necessary to decouple ionization conditions from chromatographic conditions. This can be achieved through µLC or supercritical-fluid chromatography (SFC) with post‑column addition of methanol and with chlorobenzene as a dopant allows radical cation formation for a wide range of analytes, including lipids, amino acids, and drug molecules. Notably, the CID of radical cations with a collision energy spread from 10 to 70 eV produces spectra that are orthogonal to [M+H]⁺ CID and closely resemble those of electron ionization (EI). This allows the use of radical cation CID spectra for EI library searches and in-silico structural elucidation tools based on electron ionization using liquid chromatography. Altogether, this enables the comprehensive screening of xenobiotics in biological samples, with identical retention times between electrospray ionization (ESI) and APPI with SFC facilitating feature cross-validation of putative molecules. The ability to fine-tune collision energies enhances the signal of ions related to structural features, such as methyl and double bond positions, and provides access to structural features absent in EI and [M+H]⁺ CID spectra. This enables the differentiation of isomeric metabolites, steroids and lipids through low-energy CID. For lipids, this permits rule-based de novo annotation of double bonds. The analysis can be automated by rule-based approaches using the R package MsRadaR using both high- and low-resolution instrumentation in targeted and untargeted experiments. Moreover, differential mobility spectrometry (DMS) of radical cations and protonated molecules exhibits different selectivity and allows the separation of electronic isomers of the same molecule as well as cannabinoid isomers. This illustrates that APPI is not only a complementary ionization technique to electrospray, but also an excellent option for the structural elucidation of isomers, metabolites, and lipids.