Two‐dimensional gel electrophoresis (2‐DE) facilitates the separation of thousands of proteins from highly complex protein mixtures and has become a central method in proteomics in recent years. In the present study, we examined the technical variability of large 2‐DE gels with respect to sample preparation, electrophoresis procedure, data acquisition, and biological variation by analyzing a disease (Huntington's disease) and control state with a commercially available software package, PROTEOMWEAVER™. Scatter plots and correlation coefficients were obtained to quantify both technical and biological variation. Even 2‐DE gels run separately in both dimensions yielded correlation coefficients around 0.88 and deviations from the mean close to 20% for low‐intensity spots. This indicates a high technical reproducibility of the 2‐DE procedure developed in our laboratory. Variability within a biological condition was low and comparable to technical variation (at least 0.87). Two‐dimensional (2‐D) gels obtained from samples of different biological conditions (health vs. disease) achieved a variability similar to intracondition and technical variability. These findings highlight the importance of multiple gel and spot‐by‐spot comparisons to identify biological significant changes. Minor errors introduced by technical and biological variation allow a comparison of all gels within a study which facilitates the tackling of complex biological problems.