The evolution of individual and total capsaicinoids content in three pepper varieties of Capsicum chinense Jacq. (‘Bode’ (B), ‘Habanero’ (H), and ‘Habanero Roxo’ (Hr)) during fruit ripening was studied. The five major capsaicinoids (nordihydrocapsaicin, capsaicin, dihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin) were extracted using ultrasound-assisted extraction and the extracts were analysed by ultra-performance liquid chromatography with fluorescence detection (UHPLC-Fl). The plants were grown in a glasshouse and sampled every 7 days until over-ripening. As expected, the results indicated that the total capsaicinoids content increases during the ripening of pepper fruits. The maximum contents of capsaicinoids were reached at different fruit development stages depending on the cultivar. The ‘Habanero Roxo’ pepper presented the greatest total capsaicinoids content (3.86 mg g⁻¹ fresh weigh, F.W.), followed by the ‘Habanero’ pepper (1.33 mg g⁻¹ F.W.) and ‘Bode’ pepper (1.00 mg g⁻¹ F.W.). In all the samples, capsaicin represented more than 80% of the total capsaicinoids content. Due to the high variability observed in the evolution of capsaicinoids content over the ripening process, this work intends to contribute to the existing knowledge on this aspect in relation to the quality of peppers.