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Abstract The fundamental molecules of life are polymers. Prominent examples include nucleic acids and proteins, both of which exhibit a large array of mechanical properties and three-dimensional shapes. The bending rigidity of individual polymers is quantified by the persistence length. The shape of a polymer, dictated by the topology of the polymer backbone, a line trace through the center of the polymer along the contour path, is also an important characteristic. Common biomolecular architectures include linear, cyclic (ring-like), and branched structures; combinations of these can also exist, as in complex polymer networks. Determination of persistence length and shape are largely informative to polymer function and stability in biological environments. Here we demonstratePersistence lengthShapePolymer (PS Poly), a near-fully automated algorithm designed to obtain polymer persistence length and shape from single molecule images obtained in physiologically relevant fluid conditions via atomic force microscopy. The algorithm, which involves image reduction via skeletonization followed by end point and branch point detection, is capable of rapidly analyzing thousands of polymers with subpixel precision. Algorithm outputs were verified by analysis of deoxyribonucleic acid, a very well characterized macromolecule. The method was further demonstrated by application to candidalysin, a recently discovered and complex virulence factor fromCandida albicans. Candidalysin forms polymers of highly variable shape and contour length and represents the first peptide toxin identified in a human fungal pathogen. PS Poly is a robust and general algorithm. It can be used to extract fundamental information about polymer backbone stiffness, shape, and more generally, polymerization mechanisms.