We developed an integrated platform for analysis of parameterized data from human disease models. We report a non‐negative blind deconvolution (NNBD) approach to quantify calcium (Ca2+) handling, beating force and contractility in human‐induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) at the single‐cell level. We employed CRISPR/Cas gene editing to introduce a dilated cardiomyopathy (DCM)‐causing mutation in troponin T (TnT), TnT‐R141W, into wild‐type control iPSCs (MUT). The NNDB‐based method enabled data parametrization, fitting and analysis in wild‐type controls versus isogenic MUT iPSC‐CMs. Of note, Cas9‐edited TnT‐R141W iPSC‐CMs revealed significantly reduced beating force and prolonged contractile event duration. The NNBD‐based platform provides an alternative framework for improved quantitation of molecular disease phenotypes and may contribute to the development of novel diagnostic tools.