The ventral nerve cord (VNC) of newly hatched C. elegans contains 22 motoneurons organized into three distinct classes: DD, DA, and DB, that show stereotypical positioning and arrangement along its length. The VNC represents a genetically tractable model to investigate mechanisms involved in neuron sorting and positioning. However, accurately and efficiently mapping and quantifying all motoneuron positions within large datasets is a major challenge. Here, we introduce VNC-Dist, a semi-automated software toolbox designed to overcome the limitations of subjective motoneuron positioning analysis in microscopy. VNC-Dist uses an annotator for neuron localization and an automated contour-based method for measuring the relative distances of neurons along the VNC based on deep learning and numerical analysis. To demonstrate the robustness and versatility of VNC-Dist, we applied it to multiple genetic mutants known to disrupt neuron positioning in the VNC. This toolbox will enable the acquisition and analysis of large datasets on neuronal positioning, thereby advancing investigations into the cellular and molecular mechanisms that control neuron positioning and arrangement in the VNC.