Online and automated sample extraction is widely used to increase the throughput and improve the quality of LC-MS/MS analysis. In this article, we review the most commonly used online sample extraction methodologies including online SPE, turbulent flow chromatography, online DBS extraction and online immunoaffinity extraction. We also review the offline automated sample extraction platforms, including custom robot scripts for the automation of individual steps during sample extraction, the robot scripts for the automation of individual assays, and the platform for integrated multiple sample extraction. The most recent developments and future trends in this area are also discussed.