The neuromuscular junction (NMJ) of larval Drosophila is widely used as a genetic model for basic neuroscience research. The presynaptic side of the NMJ is formed by axon terminals of motor neurons, the soma of which reside in the ventral ganglion of the central nervous system (CNS). Here we describe a streamlined protocol for dissection and immunostaining of the Drosophila CNS and NMJ that allows processing of multiple genotypes within a single staining tube. We also present a computer script called Automated Image Analysis with Background Subtraction which facilitates identification of motor nuclei, quantification of pixel intensity, and background subtraction. Together, these techniques provide a pipeline for neuroscientists to compare levels of different biomolecules in motor nuclei. We conclude that these methods should be adaptable to a variety of different cell and tissue types for the improvement of efficiency, reproducibility, and throughput during data quantification.