Dendritic spines act as sites of excitatory neuronal input in many types of neurons. Spine shape correlates with the strength and maturity of synaptic contacts. Thus, evaluation of spine morphology is relevant for studies on neuronal development, for determination of morphological correlates of learning and memory, and for analysis of mechanisms of neurodegeneration. Here, we describe a method to determine spine morphology in an ex vivo model of organotypic hippocampal slice cultures prepared from transgenic or non-transgenic mice. Spines are imaged using confocal high-resolution imaging and evaluated by algorithm-based analysis. The approach permits semiautomated determination of spine density and classification of different spine types in dendritic segments from hippocampal subregions to evaluate intrahippocampal connectivity.