Calcium/calmodulin-dependent protein kinase type II alpha (CaMKIIa) is known to play a role in both structural and functional correlates of plasticity such as learning, memory, and changes in dendritic spine size. Most studies investigating the role of CaMKIIa have been performed in vitro using tetanic stimulation protocols which are well outside the range of physiological activity occurring in the intact brain, while studies showing a requirement for CaMKIIa in cortical plasticity have been performed in knockout mice. For this reason we used an engineered FRET probe (Camuia), packaged into a herpes simplex virus (HSV), to look at changes in CaMKIIa activation that occur in vivo with physiological changes in activity. HSV-Camuia was intracortically injected into the visual cortex of ferrets during the critical period, a cranial window was implanted and chronic in vivo 2-photon imaging combined with optical imaging for determination of ocular dominance was performed. Our results revealed that changing visual cortical activity via short periods (4 hours) of monocular deprivation (MD) leads to an increase in CaMKIIa activation in spines and dendrites of cells in deprived eye dominated domains, a decrease in open eye dominated domains and no significant change relative to pre-MD levels in binocular regions of the cortex. Control experiments using a T305D/T306D mutant which renders CaMKIIa unable to bind calmodulin and therefore inactive, or sham MD experiments, showed no significant change in CaMKIIa activation. These results were further supported using western blot analysis of visual cortex contralateral or ipsilateral to the deprived eye. To date, literature investigating activity dependent changes in CaMKIIa activation have shown that large increases in neuronal activity, and calcium influx, via tetanic stimulation result in an increase in CaMKIIa activation. In contrast, our results show an increase in CaMKIIa activation in areas of the visual cortex where calcium influx is likely to be decreased due to a decrease in visual cortical drive resulting from MD. Our findings suggest that the activation/deactivation dynamics of CaMKIIa within physiological ranges of activity differ markedly from those seen at artificially induced ranges.