There is a paradigm shift in biomanufacturing towards continuous bioprocessing but cell‐based manufacturing using adherent and suspension cultures including microcarriers, hydrogel microparticles and 3D cell aggregates remains challenging due to the lack of efficient in‐line bioprocess monitoring and cell harvesting tools. Herein, we report a novel label‐free microfluidic platform for high throughput (∼ 50 particles/sec) impedance bioanalysis of biomass, cell viability and stem cell differentiation at single particle resolution. The device is integrated with a real‐time piezo‐actuated particle sorter based on user‐defined multi‐frequency impedance signatures. We first performed biomass profiling of Cytodex‐3 microcarriers seeded with adipose‐derived mesenchymal stem cells (ADSCs) to sort well‐seeded or confluent microcarriers for downstream culture or harvesting, respectively. Next, we demonstrated impedance‐based isolation of microcarriers with osteogenic differentiated ADSCs which was validated with a two‐fold increase of calcium content in sorted ADSCs. Impedance profiling of heterogenous ADSCs‐encapsulated hydrogel (alginate) microparticles and 3D ADSC aggregate mixtures was also performed to sort particles with high biomass and cell viability to improve cell quality. Overall, the scalable microfluidic platform technology enables in‐line sample processing from bioreactors directly and automated analysis of cell quality attributes to maximize cell yield and improve the control of cell quality in continuous cell‐based manufacturing.This article is protected by copyright. All rights reserved