Within the last decade, the field of proteomics has benefited biological research as well as, the pharmaceutical, food and biotech industries. The analysis pipeline is based in a bottom up approach, where proteins are digested into peptides using trypsin as enzyme, and the resulting peptides are then analyzed by liquid chromatography (LC)-mass spectrometry (MS). However, the enzymatic digestion is one of the most time-consuming steps in the analysis workflow as it is typically carried out manually. We present an efficient on-line digestion system that reduces the number of sample manipulation steps for high throughput proteomics. By incorporating a pressurized sample loop into a liquid chromatography-based separation system, both sample and enzyme (e.g., trypsin) can be simultaneously introduced to produce a complete, an ultra-fast digestion. Both standard proteins and a complex Shewanella oneidensis global protein extract were digested and analyzed using the automated on-line pressurized digestion system coupled to a mass spectrometer. The system denatured, digested, and separated product peptides in a manner of minutes, making it amenable to high-throughput applications. The on-line digestion system offers a powerful approach for high-throughput screening of proteins that could prove valuable in biochemical research; for example, quick screening of protein-based drugs.