A system is described for the on-line extraction of a digested protein in a gel spot. The extract obtained was on-line transferred to a precolumn, used to desalinate and preconcentrate the sample. The precolumn was switched in-line with an LC-ESI-MS system to separate the digest products prior to detection. By using this approach sample losses were minimized and the sample was quantitatively transferred. The benefits of the system were shown for the in-gel tryptic digest of several model proteins. Using this system, the sensitivity was 13-fold increased compared to the off-line extraction of in-gel digest products leading to a higher sequence coverage and thus a better identification potential. Also, the within day repeatability was a factor 2.5 improvement using the on-line procedure compared to the off-line extraction procedure. © 2005 Friedr. Vieweg & Sohn/GWV Fachverlage GmbH.