Mini-hydrocyclones are gaining popularity as effective tools for cell retention in perfusion cultivation. As the most widely used mammalian cell for cultivation, Chinese hamster ovary (CHO) cells experience a decline in viability after passing through mini-hydrocyclones. The reasons behind the cell viability loss within mini-hydrocyclones are not understood. This study aims to comprehensively examine the cell viability loss in relation to shear stress and pressure drop through a combination of experimental analysis and numerical simulations. The results show that a larger hydrocyclone conical factor or spigot diameter decreases pressure drop and shear stress, resulting in a lower cell viability loss. To evaluate the dependence of cell viability loss on shear stress and pressure drop, a correlation model is developed and a sensitivity analysis is performed. The results show that mean shear stress has the greatest impact on cell viability loss, followed by pressure drop and maximum shear stress.