Terpenoids are natural products with an important pharmacological interest, which are present in a number of medicinal plants. The species of Sideritis genus are valuable due to their high content in those compounds and they have been used in the Mediterranean area in folk medicine as anti-inflammatory and anti-ulcer agents. The present study describes a gradient elution reversed-phase method that uses diode array detection to determine ten pharmacologically active diterpenoids occurring in 12 species of Sideritis. First, we studied the chromatographic behaviour of standard diterpenoids to analyse the variation on retention time and the chromatographic properties with the mobile phase. Standard calibration curves were generated by plotting the area of peaks against a concentration range of the compounds. Second, the validated method was applied to the analyses of hexanic and methanolic extracts from 12 species of Sideritis, which were collected from different areas of Spain. Finally, we established for this plant a relationship between their use in folk medicine and their diterpenoid content.