Abstract A simple, economical and effective non-aqueous micellar electrokinetic chromatography (NAMEKC) method was developed for simultaneous assay of three bioactive components (cryptotanshinone, tanshinone IIA and tanshinone I) in radix Salviae miltiorrhizae and its medicinal preparations for the first time. After optimization of separation conditions, a buffer of 140 mmol l −1 sodium cholate (SC) in methanol was selected for the separation of the three tanshinones, but baseline separation of tanshinone I and tanshinone IIA in practical samples was not achieved. Therefore, second-order derivative electropherograms were applied for resolving overlapping peaks. Regression equations revealed good linear relationships (correlation coefficients 0.995–0.999) between peak heights in second-order derivative electropherograms and concentrations of the three analytes. The recoveries of three constituents ranged from 91.3 to 105.7%. The results indicated that baseline separation of the analytes was hard to be achieved in practical samples sometimes and second-order derivative electropherograms was applicable for the resolving and analysis of overlapping peaks.