The natural compound library is the most productive source in drug discovery and alkaloids are the most potential drug leads in natural compounds. Presently, systematic purification of alkaloids remains a substantial challenge. In this study, we developed an offline preparative three-dimensional HPLC (3D-HPLC) method to resolve the problem of systematic purification of alkaloids. Ten Gelsemium standards were used in the construction of the method to evaluate several factors, including column selectivity, column loadability and separation orthogonality. The offline 3D-HPLC method achieved great orthogonal selectivity and resolution power using different stationary phases, mobile phases at different pH, and different mobile phase additives. Application of this 3D-HPLC method to Gelsemium elegans Benth. was evaluated, and 24 indole alkaloids were finally isolated, including four new alkaloids and one first-identified in this plant. Moreover, a total of 229 compounds were estimated to be obtained in this plant, almost twice the number of known Gelsemium alkaloids. Therefore, this 3D-HPLC method will be efficient for systematic purification of alkaloids from Gelsemium elegans Benth. and has the potential for alkaloid preparation from other plants. CI - Copyright (c) 2021 Elsevier B.V. All rights reserved.