The rapidly growing global population and the socioeconomic shifts increased the demand for plant proteins. Notably, lupine is an underutilized legume crop that is nutritious and recognized for its high crude protein (CP). Therefore, this study was conducted to optimize the pH of protein isolate extraction (EpH) and isoelectric precipitation (IEPpH) in lupine genotypes using a central composite design (CCD). The results indicate significant variations in the chemical composition of lupine genotypes, including moisture content (6.01–7.97 g/100 g), CP (39.9–46.1 g/100 g), ether extract (6.26–13.3 g/100 g), essential amino acid index (EAAI) (62.61–70.7%), and total alkaloid (TA) (1.76–11.1%). Vitabor exhibited its maximum protein isolate purity (PIP) of 92.1% at an EpH of 8.5 and an IEPpH of 4.5. Vitabor achieved a superior protein isolate yield (PIY) of 29.0%, while the local maximum recovery (PIR) was 57.5%. All genotypes show the lowest TA at an EpH of 10 and an IEPpH of 5. The optimized extraction conditions for achieving high yield and desirable chemical compositions were as follows: EpH and IEPpH values of (10, 5) for the local, (9.86, 4.66) for Vitabor, (10, 4.69) for Sanabor, and (9.58, 4.64) for Probor. Under optimized conditions, Vitabor can serve as a sustainable source of protein isolate for low-income countries, offering high CP, EAAI, PIP, PIY, PIR, and the safest limit of TA. Meanwhile, the local and Probor are suitable for food formulations and therapeutics. Further work on the characterization of techno-functional and molecular properties is required.