Electrophoretic displays (EPDs) have excellent paper-like display features, but their response speed is as long as hundreds of milliseconds. This is particularly important when optimizing the driving waveform for improving the response speed. Hence, a driving waveform design based on the optimization of particle activation was proposed by analyzing the electrophoresis performance of particles in EPD pixels. The particle activation in the driving waveform was divided into two phases: the improving particle activity phase and the uniform reference grayscale phase. First, according to the motion characteristics of particles in improving the particle activity phase, the real-time EPD brightness value can be obtained by an optical testing device. Secondly, the derivative of the EPD brightness curve was used to obtain the inflection point, and the inflection point was used as the duration of improving particle activity phase. Thirdly, the brightness curve of the uniform reference grayscale phase was studied to set the driving duration for obtaining a white reference grayscale. Finally, a set of four-level grayscale driving waveform was designed and validated in a commercial E-ink EPD. The experimental results showed that the proposed driving waveform can cause a reduction by 180 ms in improving particle activity phase and 120 ms in uniform reference grayscale phase effectively, and a unified reference grayscale can be achieved in uniform reference grayscale phase at the same time.