The high protein content of several microalgal species makes them attractive and unconventional candidates for use in the food and pharmaceutical industries. Due to the robust cell walls of microalgae, cell rupture is necessary to improve the extraction of intracellular proteins. Thus, choosing a suitable cell-breaking treatment before protein extraction is a vital downstream processing step. Additionally, it is necessary to use an effective technique for monitoring and measuring the impact of rupture treatments on microalgal cell walls. In our study, Tetradesmus obliquus cells were disrupted using three different mechanical rupture methods: high-pressure homogenization (HPH), ultrasound (US), and ball milling (BM). The ruptured biomass cells were counted, and soluble proteins were extracted and quantified. The cell-counting technique did not detect any differences between intact and damaged cells after BM treatment because the dye (erythrosine B) did not permeate the microalgal biomass accurately. The US treatment promoted the highest yield of total protein extraction (19.95%), while the highest yields in the HPH and BM treatments were 15.68% and 14.11%, respectively. Since the cell breakage method affects protein extraction from microalgal biomass, protein release can be used as a central indicator of the degree of cell disruption.