Abstract Direct analysis of alkaloids in the tissues of crude and processed Strychnos nux-vomica seeds by MALDI-TOFMS was described. The alkaloid profiles of the herb drugs were obtained without the need of complicated sample preparation to avoid potential damage or change of the active components. Seed tissues that were optimally sliced to a thickness of 10–20 μm from the crude and processed Strychnos nux-vomica seeds as well as various parts of tissue such as endosperm and epidermis were analyzed on MALDI target plate after the matrix was directly applied onto the tissue surface. The obtained alkaloid profiles provided valuable information for the differentiation of crude and processed Strychnos nux-vomica seeds and for the explanation of the significantly different toxicity. Experimental results indicated that the direct MALDI-TOFMS analysis allowed rapid screening of the alkaloid components in Strychnos nux-vomica seeds.