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Abstract Cellular biophysical metrics exhibit systematic alterations during processes, such as metastasis and immune cell activation, which can be used to identify and separate live cell subpopulations for targeting drug screening. Image‐based biophysical cytometry under extensional flows can accurately quantify cell deformability based on cell shape alterations but needs extensive image reconstruction, which limits its inline utilization to activate cell sorting. Impedance cytometry can measure these cell shape alterations based on electric field screening, while its frequency response offers functional information on cell viability and interior structure, which are difficult to discern by imaging. Furthermore, 1‐D temporal impedance signal trains exhibit characteristic shapes that can be rapidly templated in near real‐time to extract single‐cell biophysical metrics to activate sorting. We present a multilayer perceptron neural network signal templating approach that utilizes raw impedance signals from cells under extensional flow, alongside its training with image metrics from corresponding cells to derive net electrical anisotropy metrics that quantify cell deformability over wide anisotropy ranges and with minimal errors from cell size distributions. Deformability and electrical physiology metrics are applied in conjunction on the same cell for multiparametric classification of live pancreatic cancer cells versus cancer associated fibroblasts using the support vector machine model.