Collagen characterization is crucial for disease diagnostics, prevention, and understanding, with growing focus on quantitative analysis at tissue and fibril levels. Numerous models have been developed to quantify structural changes in collagen linked to various pathologies. However, many approaches remain limited to conceptual descriptions or rely on custom software, often requiring programming skills, which restricts their clinical application and potential impact. Here we introduce CollagenFitJ, a plugin for the open-source software platform ImageJ/FIJI which represents a widely used microscopy image analysis tools. CollagenFitJ makes use of the cylindrical symmetry model for collagen to enable facile quantitative assessment of polarization-resolved second harmonic generation microscopy image stacks. The plugin’s main outputs are collagen structure-related maps (e.g., orientation and anisotropy of collagen fibrils within the focal volume), which can be accompanied by distribution and randomness maps for a series of structure-related parameters. We describe and validate the use of CollagenFitJ on images acquired on rat-tail tendon, collagen capsules surrounding human thyroid nodules and mouse colon tumors, by using both scanning and widefield second harmonic generation microscopy datasets. The plugin was designed to be user-friendly, requiring little to no experience in image processing and coding to facilitate access for life scientists and medical staff.