Capsicum fruit, a popular spice as chili pepper, is an important ingredient of the formulations used in traditional medicines. Moreover, Capsicum fruit is listed as an official drug in several pharmacopoeias. Capsaicin, the most abundant component in Capsicum fruit, exhibits its therapeutic and adverse effects in a dose-dependent manner. Therefore, the known capsaicin content is the prerequisite for optimizing any formulation based on Capsicum fruit as a crude drug. We studied 16 samples of Capsicum fruits grown at different altitudes in Nepal and determined their capsaicin content by high-performance liquid chromatography. The capsaicin content was found to range from 2.19 to 19.73 mg/g of dry weight of Capsicum fruits. Capsaicin content in pericarp was found to be higher than in seeds. No correlation was found between the shape or size of the fruits and its capsaicin content. Our findings indicate that many of the formulations prepared from Capsicum fruit, even as described in pharmacopoeias, may vary in their strength, therapeutic activity, and possible side effects if the capsaicin content in Capsicum fruit is not standardized.