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The recent push toward understanding an individual cell's behavior and identifying cellular heterogeneity has created an unmet need for technologies that can probe live cells at the single-cell level. Cells within a population are known to exhibit heterogeneous responses to environmental cues. These differences can lead to varied cellular states, behavior, and responses to therapeutics. Techniques are needed that are not only capable of processing and analyzing cellular populations at the single cell level, but also have the ability to isolate specific cell populations from a complex sample at high throughputs. The new CellMag-Coalesce-Attract-Resegment Wash (CellMag-CARWash) system combines positive magnetic selection with droplet microfluidic devices to isolate cells of interest from a mixture with >93% purity and incorporate treatments within individual droplets to observe single cell biological responses. This workflow is shown to be capable of probing the single cell extracellular vesicle (EV) secretion of MCF7 GFP cells. This article reports the first measurement of β-Estradiol's effect on EV secretion from MCF7 cells at the single cell level. Single cell processing revealed that MCF7 GFP cells possess a heterogeneous response to β-Estradiol stimulation with a 1.8-fold increase relative to the control., (© 2024 The Authors. Advanced Biology published by Wiley‐VCH GmbH.)