We report a novel method of diffraction imaging flow cytometry to measure and analyze size distribution of microspheres. An automated and robust image processing software based on the short-time-Fourier-transform algorithm has been developed to analyze the characteristic and spatially varying oscillations of side scatters recorded as a diffraction image. Our results demonstrate that the new method allows accurate and rapid determination of single microspheres' diameters ranging from 1 to 100 μm. The capacity for analysis of light scattering by two-sphere aggregates has been demonstrated but analytical tools for characterization of aggregates by multiple microspheres remain to be developed.