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Abstract. Microalgae can accumulate high levels of biomolecules that have numerous direct food and feed applications or can be transformed into a wide spectrum of high-value products for the chemical, biofuel, cosmetic, nutraceutical, and pharmaceutical industries. However, biomolecules are produced intracellularly and recovery requires cell wall lysis followed by extraction from intracellular compartments. Our prior work included development of a biological-based (enzyme-mediated) extraction and fractionation processing method for biorefining microalgae (Chlamydomonas reinhardtii) for recovery of protein and lipids into separate process streams. During process development, some proteins were released with cell lysis, and it was desired to maximize protein extraction prior to enzyme application for lipid extraction. To enhance protein solubilization after lysis, a design of experiments approach was used to evaluate the effect of pH, time, and temperature. The results were used to develop a regression model capable of determining optimized conditions for native protein solubilization. The resultant model was statistically significant and indicated that linear effects of pH, time, and temperature along with the quadratic effects of pH, and interactive effects of pH and time and pH and temperature all positively contributed to protein solubilization. Optimal conditions of pH 12, 4 h, and 45°C were determined using the model and then validated along with industrially-relevant conditions and the predicative capabilities of the model were confirmed. Following model determination and validation, the effect protein solubilization on concomitant lipid release will be explored along with protein concentration methods.