Background: Many methods are available for the concentration of proteins; however, most are not easily scalable due to costs, the need of specialized instruments and skilled workers or are very time-consuming. Three-phase partitioning (TPP) is a separation technique that has gained a lot of interest due to its rapid, simple and scalable use for concentration, isolation and decontamination of proteins from crude samples with high recovery yields. In the present work, the effect of various parameters of TPP was evaluated to optimize the concentration of proteins from Chlorella pyrenoidosa (CP), is green algae that increasingly being used as food supplements because of its positive impacts on human health. Results:Chlorella pyrenoidosa was cultivated in a closed system under controlled conditions. After reaching maximum growth, the microalgae was harvested, dried and powdered. Afterwards, TPP of CP cell lysate was done to concentrate protein content. To maximize protein concentration, various parameters were optimized such as solvent (t-butanol), ammonium sulphate concentration (40 % w/v), solid load (0.75 g/20 mL), pH (6), incubation time (20 min), slurry to butanol ratio (1:1.5) and enzymatic treatment (combination of Stargen ™ and Carezyme ™ ). Also, total starch, cellulose and carbohydrate content before and after the enzymatic treatment were determined to comprehend the impact of enzymatic treatment on protein concentration. Using these optimized parameters, 78.1 % w/w protein concentration was obtained in middle protein concentrate phase. This protein concentrate was characterizedfor proximate composition, colour analysis, water holding capacity, oil-holding capacity, foaming capacity, foam stability, amino acid composition, protein quality and thermal properties. Conclusion:Various process parameters of TPP influence the protein concentration of middle protein concentrate phase. Enzymatically treated biomass also enhanced protein concentration in middle protein concentrate phase. Characterization of protein concentrate revealed the presence high-quality protein. Therefore, it is possible to implement TPP at an industrial scale for protein concentration.