We have verified each step of in-gel digestion. We reduced the sample processing time and labor by optimizing the following four points. 1. Using intact gel pieces. 2. Use 96-well filter and positive pressure manifold before digestion. 3. Extract digested peptide without acetonitrile 4. In order to minimize non-specific absorption, digest in a low-peptides-binding microtube not in the 96-well filter or low-peptides-binding 96-well multiplate.