In this study, the evaluation of the performance of the split internal loop photobioreactor for culturing a species of green microalgae, Scenedesmus sp. under different operating superficial gas velocity and during a different time of growth (i.e., starting for the first day until end day of the culturing process) was addressed. The evaluation of the performance of the split internal loop photobioreactor was included assessing the density, pH, temperature, viscosity, surface tension, the optical density, cell population, dry biomass, and chlorophyll of the culture medium of the microalgae culturing. Additionally, the hydrodynamics of a Split Internal-Loop Photobioreactor with microalgae culturing was comprehensively quantified. Radioactive particle tracking (RPT) and gamma-ray computed tomography (CT) techniques were applied for the first time to quantify and address the influence of microalgae culture on the hydrodynamic parameters. The hydrodynamics parameters such as local liquid velocity field, shear stresses, turbulent kinetic energy, and local gas holdup profiles were measured at different superficial gas velocities as well as under different times of algae growth. The obtained results indicate that the flow distribution may significantly affect the performance of the photobioreactor, which may have substantial effects on the cultivation process. The obtained experimental data can serve as benchmark data for the evaluation and validation of computational fluid dynamics (CFD) codes and their closures. This, in turn, allows us to develop efficient reactors and consequently improving the productivity and selectivity of these photobioreactors.