Pequi (Caryocar brasiliense Camb.) is a fruit native to the Brazilian Cerrado with significant economic value. Its processing generates by-products often underused, such as its protein-rich almond. This study aimed to optimize and compare protein isolates obtained from defatted pequi almond cake using ultrasound-assisted extraction (UAE) and alkaline extraction (AE). The alkaline pH for protein solubilization was set at 9.5, and the isoelectric precipitation point at pH 4.8. UAE was optimized using a central composite design, with 56 W acoustic power, a solid-to-solvent ratio of 1/15.13 (g/mL), and extraction time of 15 min. UAE resulted in a yield of 47.25 % and protein recovery of 84.28 %, while AE achieved 41.35 % and 70.18 %, respectively. The protein isolate from UAE showed better functional properties, including higher foaming capacity and water absorption. In conclusion, UAE is a promising method for producing protein isolates, offering higher yield, shorter extraction time, and improved functional characteristics.