Context: A classic traditional Chinese medicine, Zanthoxylum nitidum (Roxb.) DC. widely used in China, exhibits anticancer, anti-inflammatory and antianalgesic activities. Alkaloids are one of the main bioactive components. It is urgent to develop a simple and reliable method to determine the main alkaloids in Z. nitidum roots. Objective: To determine the three alkaloids in Z. nitidum roots, a reversed-phase liquid chromatographic (RP-LC) method combined with an optimum extraction condition was established. Materials and methods: A method involving four-factor-three-level orthogonal array design including the extracting solvent and the RP-LC condition was assayed. Twenty batches were collected from different areas of the Guangxi Province at different harvesting times. The determined alkaloids were nitidine chloride (NC, 1), ethoxychelerythrine (2) and liriodenine (3). The stable mobile phase was a C18 packing, and the mobile phase was acetonitril-aqueous phosphoric acid-triethylamine-buffer solution. Results: The optimum extraction and detection conditions have been determined in the process of quantification of Z. nitidum root alkaloids. The three alkaloids were detected simultaneously in the 20 batches of samples. The results clearly showed that alkaloid concentrations differed significantly among Z. nitidum collected from various collection areas. Discussion and conclusion: We have established an optimum extraction and detection conditions in the process of quantification the three alkaloids in Z. nitidum roots. From this research, the most influenced factor on Z. nitidum roots was the collecting location, and the next factor was the harvesting time. The collecting location and the harvesting time should be considered as the high-quality medicinal herbs factors. © 2014 Informa Healthcare USA, Inc. All rights reserved.