Two-dimensional gel electrophoresis in combination with mass spectrometry constitutes the backbone of proteomic analysis. With the availability of powerful software tools addressing the specific needs for analyzing twodimensional gels, several typical procedures have been elaborated. In the first part of this review, we will describe and discuss the procedure of analyzing two-dimensional electrophoretic gels consisting of (i) digitizing the gels, (ii) detecting and separating individual spots, (iii) background subtraction, (iv) creating a reference gel and (v) matching the spots to the reference gel, (vi) modifying the reference gel, (vii) normalizing the gel measurements for comparison, and (viii) calibrating for isoelectric point and molecular weight markers. In the next step, (ix) a database containing the measurement results is constructed and (x) data are compared by statistical and bioinformatic means. We compare the software currently available for performing these tasks in the light of recent benchmarking and standardization efforts. We also comment on the statistical means provided in the programs including t-test statistics, ANOVA, and additional software for comparing expression patterns in large gel datasets, including hierarchical clustering algorithms and selforganizing maps (SOMs).