Digital holography has been reported as an effective tool for particle analysis. Other image-based techniques have small depth of focus allowing only 2D analysis at microscopic level. On the other hand, digital holography offers the ability to study volume samples from a single recording as reconstructions at different depths can be obtained. This paper focuses on the processing of the digital hologram that follows its recording in order to obtain particle size. We present a stepwise processing procedure with discussion on aspects such as reconstruction, background correction, segmentation, focusing, magnification and particles' feature extraction. Solutions to common obstacles faced during particle analysis which include ways to obtain fixed size reconstructions, automatically determine the threshold value, calculate magnification, and locate particles' depth position using effective focusing metrics are highlighted. Real holograms of microparticles are used to illustrate and explain the different stages of the procedure. Experimental results show that the proposed algorithm can effectively extract particle size information from recorded digital holograms.© (2008) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.