<jats:title>Abstract</jats:title><jats:p>A simple and rapid method for the simultaneous determination of seven isoquinoline alkaloids, protopine, chelidonine, coptisine, stylopine, sanguinarine, berberine, and chelerythrine, in <jats:italic>Chelidonium majus</jats:italic> L. (<jats:italic>Ch. majus</jats:italic>) samples by ultra‐performance LC method with photodiode array detection is described. The baseline separation of these compounds was performed with (A) acetonitrile–(B) ammonium acetate (10 mM, adjusted to pH 3.0 with acetic acid) as the mobile phase using a C18 RP column (2.1×100 mm, 1.7 μm). Optimized conditions resulted in excellent peak shapes. The seven alkaloids were completely separated within 20 min. Good linear behaviors (<jats:italic>r</jats:italic>≥0.9992) over the investigated concentration ranges were observed for all the analytes. Validation proved the repeatability of the method was good and recovery was satisfactory. The validated method was successfully applied for 20 batches of <jats:italic>Ch. majus</jats:italic>. These results demonstrated that the ultra‐performance LC photodiode array method proposed was very useful in the analysis and quality control of <jats:italic>Ch. majus</jats:italic>.