Abstract— In this paper, a new serial algorithm for highspeed autofocusing of cells is proposed. The proposed algorithm extracts depth information of target cells from their diffraction patterns. High-speed focusing of yeast cells with 20 ms response time was demonstrated. Continuous autofocusing of yeast cells with a scanning microscope was also demonstrated. The successful continuous autofocusing suggests that image-based highthroughput measurement of cells could be realized using the proposed algorithm.