This study contributes to the investigation of alkaloids from Strychnos usambarensis. It includes botanical and ethnobotanical analysis of the plant material, optimization of extraction methods (such as lyophilization instead of vacuum concentration to preserve sensitive alkaloids and modifications to chromatographic techniques), and structural elucidation of isolated alkaloids using spectroscopic methods (UV, IR, NMR, and mass spectrometry). Key findings are the isolation of four new asymmetric dimeric indole alkaloids: usambarensine (C₂₉H₂₈N₄), 3-4 dihydro-usambarensine (C₂₈H₃₀N₄), N'b-methyl-usambarensine, and N'b-methyl-3-4 dihydro-usambarensine. These alkaloids are characterized by a harmane or 3-4 dihydro-harmane moiety combined with a tetrahydro-β-carboline fragment, representing a new type of Strychnos alkaloids distinct from previously reported symmetric dimeric quaternary alkaloids. Chemotaxonomic analysis classifies these alkaloids within the Strychnos-type indole alkaloids, showing structural similarities to compounds from Apocynaceae (e.g., Vinca, Alstonia) and Rubiaceae. The study also addresses extraction challenges, proposes a lyophilization-based protocol to prevent degradation, and highlights African Strychnos species as a source of novel alkaloids with potential pharmacological activities (e.g., curarizing, hallucinogenic, hypotensive).