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Neurons, due to their long axons and dendrites, are particularly dependent on proper intracellular trafficking and vulnerable to defects in the movement of intracellular cargo that are noted in neurodegenerative and neurodevelopmental disorders. Accurate quantification of intracellular transport is therefore needed for studying the mechanisms of cargo trafficking, the influence of mutations, and the effects of potentially therapeutic pharmaceuticals. In this article, we introduce an algorithm called \u201cKymolyzer.\u201d The algorithm can quantify intracellular trafficking along a defined path, such as that formed by the aligned microtubules of axons and dendrites. Kymolyzer works as a semi\u2010autonomous kymography software application. It constructs and analyzes kymographs to measure the movement and distribution of fluorescently tagged objects along a user\u2010defined path. The algorithm can be used under a wide variety of experimental conditions and can extract a diverse array of motility parameters describing intracellular movement, including time spent in motion, percentage of objects in motion, percentage of objects that are stationary, and velocities of motile objects. This article serves as a user manual describing the design of Kymolyzer, providing a stepwise protocol for its use and illustrating its functions with common examples. \u00a9 2020 Wiley Periodicals LLC<\/jats:p><jats:p><jats:bold>Basic Protocol<\/jats:bold>: Kymolyzer, a semi\u2010autonomous kymography tool to analyze intracellular motility<\/jats:p>","DOI":"10.1002\/cpcb.107","type":"journal-article","created":{"date-parts":[[2020,6,12]],"date-time":"2020-06-12T14:55:48Z","timestamp":1591973748000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":30,"title":["Kymolyzer, a Semi\u2010Autonomous Kymography Tool to Analyze Intracellular Motility"],"prefix":"10.1002","volume":"87","author":[{"given":"Himanish","family":"Basu","sequence":"first","affiliation":[{"name":"Kirby Neurobiology Center Boston Children's Hospital  Boston Massachusetts"},{"name":"Division of Medical Sciences Harvard Medical School  Boston 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