Spoti-find: A novel, open-source void spot assay image analysis tool

The void spot assay (VSA) is a widely used, non-invasive method for evaluating urinary behavior in rodents, but existing analysis tools are limited in scope, throughput, or accessibility. We developed Spoti-find, a stand-alone, open-source VSA image analysis application that introduces novel, biologically meaningful metrics including void circularity, distance to paper edge, and volume-based binning into primary, micro-, and nanovoids. Designed with usability in mind, Spoti-find features a graphical interface that enables manual or semi-automated spot identification, adjustable thresholds, and streamlined data export without the need for coding expertise. We validated Spoti-find across diverse datasets, showing strong inter-user consistency, sensitivity to known phenotypes in aging and disease models, and accuracy in capturing novel parameters while demonstrating high agreement with existing tools. By capturing behavioral context and spatial morphology in voiding patterns, Spoti-find expands the interpretive power of VSA and provides a flexible, user-friendly platform for phenotyping urinary dysfunction in preclinical studies.