The use of high‐throughput flow cytometry to characterize nanoparticles has received increased interest in recent years. However, to fully realize the potential of flow cytometry for the characterization of nanometer‐sized objects, suitable calibrators for size estimation must be developed and the sensitivity of conventional flow cytometers has to be advanced. Based on the scattered signal, silica and plastic beads have often been used as flow cytometric size calibrators to evaluate the size of extracellular vesicles and artificial vesicles (liposomes). However, several studies have shown that these beads are unable to accurately correlate scatter intensity to vesicle size. In this work, we present a novel method to estimate the size of individual liposomes in flow cytometry based on liposomal size calibrators prepared by fluorescence‐activated cell sorting (FACS), here coined fluorescence‐activated nanoparticle sorting (FANS). These calibration liposomes exhibit sizes, structures, and refractive indexes identical to the particles being studied and thus can serve as unique calibrators. First, a sample of polydisperse fluorophore‐labeled unilamellar liposomes was prepared and analyzed by flow cytometry. Next, different fractions of the polydisperse liposomes were FANS‐sorted according to their fluorescence intensity. Thereafter, we employed nanoparticle tracking analysis (NTA) to evaluate the liposome sizes of the FANS‐sorted liposome fractions. Finally, we correlated the flow cytometric readouts (side scatter and fluorescence intensity) of the FANS‐sorted liposome fractions with their corresponding size obtained by NTA. This procedure enabled us to translate the liposome fluorescence intensity to the liposome size in nanometers for all detected individual liposomes. We validated the size distribution of our polydisperse liposome sample obtained from flow cytometry in combination with our FANS‐calibrators against standard methods for sizing nanoparticles, including NTA and cryo‐transmission electron microscopy. This work also highlights the limitation of using the flow cytometric side scattering readout to determine the size of small (30–300 nm) artificial vesicles. © 2019 International Society for Advancement of Cytometry