Sensory neurons in the vomeronasal organ (VNO) are thought to mediate a specialized olfactory response. Currently, very little is known about the identity of stimulating ligands or their cognate receptors that initiate neural activation. Each sensory neuron is thought to express 1 of approximately 250 variants of Vmn1Rs, Vmn2Rs (A, B, or D), or FPRs which enables it to be tuned to a subset of ligands (Touhara and Vosshall, Annu Rev Physiol 71:307-332, 2009). The logic of how different sources of native odors or purified ligands are detected by this complex sensory repertoire remains mostly unknown. Here, we describe a method to compare and analyze the response of VNO sensory neurons to multiple stimuli using conventional calcium imaging. This method differs from other olfactory imaging approaches in that we dissociate the tightly packed sensory epithelium into individual single cells. The advantages of this approach include (1) the use of a relatively simple approach and inexpensive microscopy, (2) comparative analysis of several hundreds of neurons to multiple stimuli with single-cell resolution, and (3) the possibility of isolating single cells of interest to further analyze by molecular biology techniques including in situ RNA hybridization, immunofluorescence, or creating single-cell cDNA libraries (Malnic et al., Cell 96:713-723, 1999).