Experimental visual pathway lesion in the form of optic nerve (ON) crush or transection injury results in massive death of retinal ganglion cells (RGCs) and permanent loss of synaptic connections (Berkelaar et al., J Neurosci 14:4368-4374, 1994). Despite the fact that RGC axon regeneration is inhibited in a manner typical of other CNS lesions, the rodent ON injury model is one of the few models where robust axon regeneration has been achieved after therapeutic intervention (Berry et al., Restor Neurol Neurosci 26:147-174, 2008). However, assessment of the efficacy of therapeutic approaches in promoting ON regeneration has traditionally relied on histological methods, which necessitate the sacrifice of experimental animals and thus preclude longitudinal in vivo monitoring of individual subjects. Manganese-enhanced MRI (MEMRI) utilizes the paramagnetic properties and uptake and transport mechanisms of manganese ions (Mn(2+)) by neurons, thus enabling serial in vivo monitoring of the entire axonal projections (Sandvig et al., J Magn Reson Imaging 34:670-675, 2011; Thuen et al., J Magn Reson Imaging 4:492-500, 2005; Pautler et al., Magn Res Med 50:33-39, 2003; Saleem et al., Neurotechnique 34:685-700, 2000). The above properties of Mn(2+) render MEMRI a highly suitable technique for assessment of ON regeneration after injury, especially with a view to in vivo monitoring of neuronal connectivity and axon-regenerative responses to treatment. In this chapter, we provide a generic protocol for ON lesioning and MEMRI application for assessment of ON regeneration in rodents.