Traumatic brain injury (TBI) is unique among neurological afflictions in that it is induced by a discrete physical event. To understand the relationship between mechanical loading and the evolution of structural and functional alterations of neural cells, TBI researchers have utilized in vitro models. These models were engineered to mimic loading conditions relevant for clinical TBI and to allow for the microscopic study of the cellular responses in real time. Collectively, this high degree of experimental control has resulted in robust platforms that enable the exploration of biological mechanisms involved in the progression of neural cellular injury. This chapter presents detailed background and methodology pertaining to two established in vitro models used in the field: (1) “stretch” injury to two-dimensional (2-D) cultures, and (2) simple shear deformation applied to three-dimensional (3-D) cell-containing matrices. The stretch injury paradigm uses a rapid pressure-pulse to stretch an elastic silicone membrane on which neural cells are cultured. The resulting deformation can be either biaxial or uniaxial, and is commonly applied to 2-D neuronal cultures with isolated axonal projections to model tensile loading in aligned axonal tracts, believed to be a proximal cause of diffuse axonal injury, the “hallmark” pathology of closed-head TBI. Rapid shear deformation to 3-D neural cellular constructs is applied using a linear actuator and is designed to replicate the complex loading conditions experienced by brain cells during inertial loading, with shear being the dominant mode of deformation in the nearly incompressible brain. This model has been utilized to study the acute and longer-term responses of 3-D neuronal cultures or 3-D neuronal-astrocytic cocultures to heterogeneous strain fields representative of loading patterns in vivo.