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

The void spot assay (VSA) is a widely used, noninvasive 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 semiautomated spot identification, adjustable thresholds, and streamlined data export without the need for coding expertise. We validated Spoti-find across diverse datasets, showing strong interuser 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.NEW & NOTEWORTHY The VSA is a popular, noninvasive method for studying rodent urinary behavior, but current tools lack flexibility and require extensive manual quality control. We developed Spoti-find, an open-source application introducing novel parameters like void circularity, edge proximity, and volume-based binning. With a user-friendly interface requiring no coding, Spoti-find enables manual and semiautomated analysis with high consistency and transparency.