The superficial dorsal horn (SDH) is a critical site for pain processing and regulation, containing subnetworks of local interneurons responsible for integration of sensory information received from peripheral afferents. Through epifluorescent microscopy on acute SDH slices, intracellular calcium increases were recorded as a marker for action potential firing at the subnetwork level. Responses within sham control animals were compared to models of inflammatory and neuropathic pain. A developed custom image processing pipeline combined MATLAB and IJM scripts with peer-reviewed toolboxes to quantify changes in SDH subnetwork excitability across pain models. The presented harmonized pipeline serves as a novel assay for specific and multidimensional evaluation of SDH circuitry. No significant differences were observed across evoked SDH subnetworks. Future experiments will use these pipeline approaches to investigate potential differences in spontaneous SDH network activity between the experimental groups, which may underlie differential pain sensitivities in these chronic pain conditions.