Abstract For the identification of proteins from biological samples by mass spectrometry there are two scenarios. Proteins are pre-fractionated in some way or are analysed as a complex mixture (shotgun). Shotgun proteomics recently became more popular, because of technological developments on the mass spectrometer side which now allows the identification of several thousand proteins from a complex biological matrix at one go. However, in many cases it is still useful to separate proteins first in a gel. But not only mass spectrometer technology made progress. Recently, new protocols and techniques which make the analysis of starting material in the low microgram range possible, and also simplify the whole procedure, were developed. In this small review detailed protocols will be described which also allow inexperienced beginners to get good results.