A novel method was proposed for simultaneous measurement of diffusion coefficients, (D), from mixed samples by electrophoresis and termed electromigration-based diffusivity spectrometry. After theoretical treatment, D-equation for practical use has been deduced. With a modified CE system built in laboratory, electromigration-based diffusivity spectrometry has been realized and validated to suit for fast and accurate determination of diffusivities of mixed aromatic amino acids, phenols and aromatic organic acid, giving diffusivity spectra by peak area versus D, much similar to mass spectra. The precision of the measurement was found to critically depend on pH value of running buffer, which should be so selected that the analytes and internal standards could be charged at above 0.5e. The standards have to be selected at an electric flux far from each other and from analytes. In these cases, sample and running buffer concentrations, voltage and system temperature were found to have only negligible impact on the determination. In our test, the obtained measuring precision was generally kept within 1% for five runs, and the measured values of D agreed well with those from literature, with a deviation of less than 2.2% after the right use of calibration standards.