The presented work investigated two biomasses Baker’s yeast (Saccharomyces cerevisiae) and microalgae (Chlorella vulgaris), through characterisation of their cell disruptions in a high-pressure homogenizer (HPH). As energy producing biomasses, emphasis has been placed on optimizing the yeast/microalgae through determining the protein concentration yields and associated cost to determine its economic feasibility. Through a One-Variable-At-a-Time (OVAT) approach the dataset range was established for the parameters. The results presented show yeast/microalgae homogenized at various pressures (30 - 90 MPa), temperature (15 - 25 °C) as well as (30 - 50 °C), and the number of cycles (passes) (1 - 5) against two responses; protein concentration yield and cost. The high-pressure homogenizer (HPH), GYB40-10S (with a two stage homogenizing valves pressure with a maximum pressure of 100 MPa) was used to cause cell disruption. The homogenate in categorical ratio to buffer solution (Solution C) of 10:90; 20:80 and 30:70 was centrifuged. Design Expert Software; Design of Experiment (DOE) was used in establishing the design matrix and to also analyse the experimental data. The relationships between the yeast/microalgae homogenizing parameters (pressure, number of cycles, temperature, and ratio) and the two responses (protein concentration and operating cost) were established. Also, the optimization capabilities in Design-Expert software were used to optimize the homogenizing process. The mathematical models developed were tested for adequacy through the analysis of variance (ANOVA) and other adequacy measures. In this investigation, the optimal homogenizing conditions were identified at a pressure of 90MPa, 5 cycles, a temperature of 20 oC and a buffer solution ratio of 30:70 which yielded a maximum protein concentration of 1.7694 mg/mL, and a minimum total operating cost of 0.28 Euro/hr for a 15 to 25 oC temperature range for Baker’s yeast (Saccharomyces cerevisiae) as biomass.