Shear stress during bioreactor cultivation has significant impact on cell health, growth, and fate. Mammalian cells, such as T cells and stem cells, in next-generation cell therapies are especially more sensitive to shear stress present in their culture environment than bacteria. Therefore, a base knowledge about the shear stress imposed by the bioprocesses is needed to optimize the process parameters and enhance cell growth and yield. However, typical computational flow dynamics modeling or PCR-based assays have several limitations. Implementing and interpreting computational modeling often requires technical specialties and also relies on many simplifications in modeling. PCR-based assays evaluating changes in gene expression involve cumbersome sample preparation with the use of advanced lab equipment and technicians, hampering rapid and straightforward assessment of shear stress. Here, we developed a simple, cell-based shear stress sensor for measuring shear stress levels in different bioreactor types and operating conditions. We engineered a CHO-DG44 cell line to make its stress sensitive promoter EGR-1 control GFP expression. Subsequently, the stressed CHO cells were transferred into a 96 well plate, and their GFP levels (population mean fluorescence) were monitored using a cell analysis instrument (Incucyte®, Sartorius Stedim Biotech) over 24 hours. After conducting sensor characterization, which included chemical induced stress and fluid shear stress, and stability investigation, we tested the shear stress sensor in the Ambr® 250 bioreactor vessels (Sartorius Stedim Biotech) with different impeller and vessel designs. The results showed that the CHO cell-based shear stress sensors expressed higher GFP levels in response to higher shear stress magnitude or exposure time. These sensors are useful tools to assess shear stress imposed by bioreactor conditions and can facilitate the design of various bioreactor vessels with a low shear stress profile.