Based on the published algorithms or tools developed by our and other groups, we introduce a detailed protocol for the most comprehensive and up-to-date genome synteny pipeline (called PanSyn) and provides step-by-step instructions as well as application examples for demonstrating how to use it. PanSyn pipeline includes three major modules (microsynteny analysis, macrosynteny analysis, and integrated micro & macro analysis). PanSyn not only fills a gap of lacking a user-friendly, highly-customized tool for genome macrosynteny analysis but also allows for integrated pan-evolutionary and regulatory analysis of genome microsyntenty and macrosynteny which are not yet available in any public synteny software or tools. PanSyn has been tested under Linux system. PanSyn has multiple subroutines. Users only need to simply modify the configuration file and corresponding command parameters to execute them. Outputs include vector diagrams that are suitable for custom modification.