Sacha Inchi (Plukenetia volubilis L.) is a plant native in the Amazon rainforest in South America known for its edible seeds, which are rich in lipids, proteins, vitamin E, polyphenols, minerals, and amino acids. Rural communities in developing nations have been using this plant for its health benefits, including as a topical cream for rejuvenating and revitalising skin and as a treatment for muscle pain and rheumatism. Although Sacha Inchi oil has been applied topically to soften skin, treat skin diseases, and heal wounds, its protein-rich seeds have not yet received proper attention for extensive investigation. Proteins in Sacha Inchi seeds are generally known to have antioxidant and antifungal activities and are extensively used nowadays in making protein-rich food alternatives worldwide. Notably, large-scale use of seed proteins has begun in nanoparticle and biofusion technologies related to the human health-benefitting sector. To extract and identify their proteins, the current study examined Sacha Inchi seeds collected from the Malaysian state of Kedah. Our analysis revealed a protein concentration of 73.8 ± 0.002 mg/g of freeze-dried seed flour. Employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) and PEAKS studio analysis, we identified 217 proteins in the seed extract, including 152 with known proteins and 65 unknown proteins. This study marks a significant step towards comprehensively investigating the protein composition of Sacha Inchi seeds and elucidating their potential applications in the food and biopharmaceutical sectors. Our discoveries not only enhance our knowledge of Sacha Inchi's nutritional characteristics but also pave the way for prospective research and innovative advancements in the realms of functional food and health-related domains.