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Here, we present an open\u2010source, scalable imaging and analysis platform that integrates optogenetic stimulation, calcium imaging, automated acquisition, single\u2010cell and network analyses. The platform enables robust quantification of spontaneous and evoked neuronal activity across hundreds of human stem cell\u2010derived neurons over multiple timepoints, supporting functional phenotyping at both cellular and network levels. We demonstrate the versatility of the platform across multiple disease\u2010relevant contexts, including models of CDKL5 Deficiency, SSADH Deficiency, and tuberous sclerosis complex (TSC). Additionally, we generate CRISPR\u2010Cas9 knock\u2010in hiPSC lines expressing GCaMP6s and demonstrate partial reversal through pharmacological intervention in TSC. By linking single\u2010cell dynamics to network\u2010level measures, this platform provides a generalizable framework for scalable functional phenotyping and high\u2010throughput screening in human neuronal models.<\/jats:p>","DOI":"10.1002\/advs.202515887","type":"journal-article","created":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T15:14:42Z","timestamp":1773069282000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["An Open\u2010Source Pipeline for Calcium Imaging and All\u2010Optical Physiology in Human Stem Cell\u2010Derived Neurons"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9488-7893","authenticated-orcid":false,"given":"Wardiya","family":"Afshar\u2010Saber","sequence":"first","affiliation":[{"name":"Department of Neurology, F.M. 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