Mixing operations in biological processes is of utmost importance due to its effect on scaling-up and heat and mass transfer. This paper presents the characterization of a bench-top bioreactor with different impeller configurations, agitation and oxygen transfer rates, using CFD simulations and experimental procedures. Here, it is demonstrated that factors such as the type of impeller and the flow regime can drastically vary the operation as in the preparation of cultures. It was observed that the bioreactor equipped with a Rushton generates a k L a of 0.0056 s−1 for an agitation velocity and airflow rate of 250 RPM and 5 L/min, respectively. It is suitable result for the dissolved oxygen (DO) but requires a considerable amount of power consumption. It is here where the importance of the agitator’s diameter can be observed, since, in the case of the two propeller types studied, lower energy consumption can be achieved with a smaller diameter, as well as a much smaller shear cup 2.376 against 0.723 s−1 by decreasing by 4 cm the standard diameter of an agitated tank (10 cm). Finally, the k L a values obtained for the different configurations are compared with the maximum shear rate values of different cell cultures to highlight the impact of this study and its applicability to different industries that use agitation processes for cell growth.