<jats:p>A simple and sensitive method was developed and validated for profiling and simultaneous quantitation of seven alkaloids (6‐hydroxy‐β‐carboline‐1‐carboxylic acid, β‐carboline‐1‐carboxylic acid, β‐carboline‐1‐propanoic acid, 3‐methylcanthin‐5,6‐dione, 5‐hydroxy‐4‐methoxycanthin‐6‐one, 1‐methoxycarbony‐β‐carboline, and 4,5‐dimethoxycanthin‐6‐one) in <jats:italic><jats:styled-content style="fixed-case">P</jats:styled-content>icrasma quassioide</jats:italic> grown in different locations by high‐performance liquid chromatography with photodiode array detection. The analysis was conducted on a <jats:styled-content style="fixed-case">P</jats:styled-content>henomenex <jats:styled-content style="fixed-case">G</jats:styled-content>emini <jats:styled-content style="fixed-case">C</jats:styled-content><jats:sub>18</jats:sub> column at 35°C with mobile phase of 25 mM aqueous ammonium acetate (p<jats:styled-content style="fixed-case">H</jats:styled-content> 4.0, adjusted by glacial acetate acid) and acetonitrile. A common fingerprint chromatograph under 254 nm consisting of 27 peaks was constructed for the evaluation of the similarities among 31 <jats:italic><jats:styled-content style="fixed-case">P</jats:styled-content>. quassioide</jats:italic> samples. Samples from <jats:styled-content style="fixed-case">G</jats:styled-content>uangdong and <jats:styled-content style="fixed-case">G</jats:styled-content>uangxi Provinces were found to be within group linkage and showed significant difference from that of <jats:styled-content style="fixed-case">J</jats:styled-content>iangxi Province origin by using principal component analysis and hierarchical clustering analysis. In addition, the seven alkaloids were identified by electrospray ionization mass spectrometry and comparing with reference standards and literature data. All of them were determined simultaneously using the established <jats:styled-content style="fixed-case">HPLC</jats:styled-content> method. This rapid and effective analytical method could be employed for quality assessment of <jats:italic><jats:styled-content style="fixed-case">P</jats:styled-content>. quassioide</jats:italic>, as well as pharmaceutical products containing this herbal material.