In this study, sixteen secondary metabolites, including two chromones, four dibenzofurans, three lipids, three depsides, two aromatic compounds, a quinone, and a terpene, were detected in the methanol:acetone (1:1 v/v) extract of the lichen Hypotrachyna enderythraea (Zahlbr.) Hale, using High-Performance Liquid Chromatography coupled to Orbitrap Electrospray Ionization tandem Mass Spectrometry (HPLC-Orbitrap ESI tandem MS/MS). These metabolites were characterized by analysis of their exact molecular masses and corresponding fragmentation patterns. The retention times of the identified metabolites were compared with those of standard compounds, confirming the presence of naturally occurring bioactive compounds. Density Functional Theory (DFT) calculations were employed to investigate preferential deprotonation sites in representative polyprotic metabolites. All these findings may contribute to expanding the spectrum of compounds identified within the genus Hypotrachyna and to evaluating their potential biological activities. Keywords: Hypotrachyna species; secondary metabolite profiling; lichen; UHPLC-MS-MS.