The reproducibility and resolving power of immobilized pH gradient (IPG) led two-dimensional gel electrophoresis (2-DE) technology to the heart of proteome projects. Despite the recent progress achieved in this field, 2-DE is still not widely used as a screening tool either in industry or in clinical chemistry laboratories. This is mostly owing to the fact that this methodology is complex, expensive, and slow. Actually, the whole process for a standard 2-DE (160×180 mm) requires three to four working days depending on which procedure is used. In order to reduce significantly the above limiting factors, a mini-2-DE method was developed that has the ability to produce several protein maps easily within one working day (<6 h) at a much lower price. It uses a combination of in-gel sample rehydration (1,2), small nonlinear 3.5–10 IPG gels (7 cm), homemade or precast vertical slab gels, and an automated protein-staining device. Examples of plasma and Escherichia coli protein separation are presented in this chapter. It demonstrates that this method is rapid, simple to perform, and keeps the advantage of the 2-D resolving power. Therefore, it opens the prospect of high 2-DE throughput for sample screening.