Flow/hyperlayer field flow fractionation (Fl/Hy FFF) has been applied for the rapid separation and size characterization of core-shell type poly(l-lactide) (PLLA) microspheres which are widely studied as a potential drug carrier due to its biodegradability. Examined microspheres are prepared by varying the entrapped amount of the retinoic acid as an anti-cancer therapeutic and varying PEG-PLLA copolymers. It is demonstrated that the separation of PLLA microspheres can be achieved within 5 min. by Fl/Hy FFF and the particle size distribution (PSD) is readily obtained from the experimental fractograms. From the size distribution of PLLA particles obtained by Fl/Fl FFF, it is shown that PSD increases with the amount of drug loaded but it does not seriously change with the addition of PEG-PLLA copolymers.