Fluorescent biosensors are important measurement tools for in vivo quantification of pH, concentrations of metal ions and other analytes, and physical parameters such as membrane potential. Both the development of these sensors and their implementation in examining cellular heterogeneity requires technology for measuring and sorting cells based on the fluorescence levels before and after chemical or physical perturbations. We developed a droplet microfluidic platform for the screening and separation of cell populations on the basis of the in vivo response of expressed fluorescence-based biosensors after addition of an exogenous analyte. We demonstrate the capability to resolve the responses of two genetically encoded Zn2+ sensors at a range of time points spanning several seconds and subsequently sort a mixed-cell population of varying ratios with high accuracy.