Cassava, predominantly cultivated for its tubers, generates leaves that are often considered agricultural waste despite their rich protein, vitamin, and mineral content. However, the presence of cyanogenic glycosides and condensed tannins limits their direct human consumption. With the rising demand for plant-based proteins driven by the increase in veganism and the need for sustainable protein sources, this study aims to valorize cassava leaves as a protein source, thereby reducing post-harvest waste. Various cassava leaf varieties were screened based on growth stage, protein content, and levels of cyanide and tannins. The mature leaves of the Shree Jaya variety, identified as optimal, underwent protein extraction via an alkaline extraction-isoelectric precipitation method. This method was optimized for maximum protein content (21.83 ± 0.41 g/100 g dm), extraction yield (18.31 ± 0.53%), and protein recovery yield (69 ± 1.31%) using Design Expert software. The optimal extraction conditions were 114 minutes, 46°C temperature, 23.5 mL/g solvent-to-solute ratio, and pH 11.0. Despite successful protein isolation, cyanide and tannin contents exceeded permissible limits, necessitating detoxification. Traditional methods revealed that drying pounded cassava leaves followed by multiple washings effectively reduced cyanide and tannin content by 85% and 69%, respectively, without compromising protein content. The detoxified cassava leaf protein isolate (DCPI) exhibited changes in compositional, structural, morphological, molecular, and thermal characteristics compared to the control. Moreover, the functional properties of the protein isolate improved after detoxification under different pH conditions, making it suitable as an active ingredient in various foods. Although the overall amino acid scores improved to 89.19% after detoxification, essential hydrophilic and pH-sensitive amino acids were reduced to 53.45% compared to the control (56.78%). Deep eutectic solvents (DES), known for their sustainability and non-toxicity, were explored for protein extraction to address this reduction and restore the original amino acid content. Various DES mixtures were characterized and compared to water, with lactic acid and glycerol identified as the most effective combination. Under the same optimized conditions, DES increased protein content to 22.16 ± 0.36% and protein recovery yield to 73.77 ± 1.05%, although the extraction yield was reduced to 16.85 ± 0.41%. The DES-assisted extraction method (DAE) enhanced the protein isolate's nutritional, structural, and functional properties and increased amino acid scores (91.24%) but yielded lower extraction volumes. Ultrasound-assisted extraction (USDAE) with DES was subsequently optimized to improve the extraction yield further. The protein content and recovery yield of the protein isolate were maximized by 40.56 ± 0.54% and 76.82 ± 1.44%, respectively, at optimal conditions of 54% of ultrasound amplitude, 34 min extraction time, 20.5 ml/g with 15% of water addition. Moreover, this method significantly improved the yield to 25.91% while maintaining superior amino acid composition (94.03%), including hydrophilic and hydrophobic amino acids. The final protein isolate demonstrated excellent functional and structural qualities, suggesting its potential as a replacement for foaming agents and emulsifiers in the bakery industry.