Pumpkin (Cucurbita moschata) is recognized as a functional food due to its rich content of nutrients and bioactive compounds with health-promoting properties. Its seeds, often discarded, are a valuable source of lipids, proteins, fiber, minerals, vitamins, carotenoids, phytosterols, tocopherols, and polyphenols. Notably, their high protein content results in a significant concentration of amino acids (AAs), essential for various metabolic and physiological processes. Roasting is a traditional cooking method that can enhance sensory attributes and improve the bioaccessibility and bioavailability of nutrients and phytochemicals, including AAs. This study aims to evaluate the impact of traditional roasting methods on the AA profile of pumpkin seeds (Cucurbita moschata), from the Cuyano INTA and Cokena INTA cultivars. Seeds were roasted in a non-stick pan (180ºC, 10 min) and an oven (180ºC, 15 min). Protein-bound AAs were extracted following AOAC method 994.12 and quantified by means of RP-UHPLC. Twenty AAs, including the essential ones, were identified. ANOVA showed significant differences in AA levels among cultivars and treatments; however, no significant interaction was observed. Cooking treatment explained 60.69% of the variation, while cultivar accounted for 15.23%, indicating that roasting method was the dominant factor. Seeds of Cuyano INTA exhibited the highest AA concentrations. For essential AAs, Cuyano INTA seeds had 1.8 times higher levels than Cokena INTA (14.77 vs. 8.39 mg g⁻¹). Roasting significantly affected the AA composition, especially affecting heat-sensitive AAs. Compared to raw seeds, roasting caused AAs losses of up to 50%. These findings demonstrate that roasting strongly influences the nutritional quality of pumpkin seeds, with potential implications for their use in functional foods and dietary applications.