Harmaline and harmine accounted for more than 70% in composition in extracts of P. harmala. More attention, however, should be paid to the other alkaloids which would be favorable or unfavorable to the efficacy and safety of the products. It was necessary to determine these trace alkaloids in the extracts; thereafter, most of them have been characterized. Diglycoside vasicine, vasicine, vasicinone, harmalol, harmol, tetrahydroharmine, 8-hydroxy-harmine, ruine, harmaline, and harmine were separated and identified with reference substances and characteristic MS spectra in extracts by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and high-performance liquid chromatography (HPLC). Three trace alkaloids, vasicine, harmalol, and harmol were determined using the developed chromatographic separation method subsequently. The average contents of vasicine, harmalol, and harmol in extracts of ten batches were 2.53 ± 0.73, 0.54 ± 0.19, and 0.077 ± 0.03%, respectively. The total content of the three alkaloids was 3.23 ± 0.90% (from 1.81 to 4.48%). For rough estimation of all the relative alkaloids except of harmaline and harmine, the average total areas of all peaks in extracts varied from 4.35 to 26.64% detected at 220, 254, 265, 280, and 380 nm, respectively. The results indicated that area normalization method was powerless for the quality evaluation for traditional herb medicine consisting of numerous compounds with highly differential features. It might be concluded that LC-MS or HPLC could be utilized as a qualitative and quantitative analytical method for quality control of the extracts from seeds of P. harmala L.