<p>Alkaloids from the <i>Strychnos</i> genus exhibit remarkable chemical diversity and are considered promising candidates for drug development. However, many endemic species remain unexplored. In this study, we report the use of a scouting liquid chromatographic system for method development and a liquid chromatography system hyphenated to high-resolution mass spectrometry (LC-HRMS) to efficiently profile and annotate the specialized metabolites of <i>Strychnos peckii</i> B.L. Rob. A structural database was employed to curate the data through MS² fragmentation experiments and the propagation of the molecular network using in silico tools. Manual data curation, based on biosynthetic pathways, comparisons with public data, and the creation of an in-house database, led to the annotation of 46 metabolites. The annotated metabolites were classified into alkaloids (33), benzoic acids (6), and flavonoids (5). Based on fragmentation patterns and their biogenesis, the alkaloids were further categorized into β-carbonyl compounds (<b>1</b>–<b>5</b>), monoterpene-indole alkaloid aglycones (<b>6</b>–<b>11</b>), monoterpene-indole alkaloids (<b>12</b>–<b>20</b>), monoterpene-indole alkaloid lactones (<b>21</b>–<b>25</b>), quinolinic compounds (<b>26</b>), and tryptophan-derived alkaloids (<b>27</b>–<b>30</b>), among others. The annotated alkaloids are well-known for their pharmacological properties. Overall, this study enhances the chemical understanding of <i>Strychnos peckii</i>.</p> Graphical Abstract <p></p>