<p>Top-down analysis of intact proteins and middle-down analysis of proteins subjected to limited digestion require efficient detection of traces of proteoforms in samples, necessitating the reduction of sample complexity by thorough pre-fractionation of the proteome components in the sample. SDS–PAGE is a simple and inexpensive high-resolution protein-separation technique widely used in biochemical and molecular biology experiments. Although its effectiveness for sample preparation in bottom-up proteomics has been proven, establishing a method for highly efficient recovery of intact proteins from the gel matrix has long been a challenge for its implementation in top-down and middle-down proteomics. As a much-awaited solution to this problem, we present an experimental protocol for efficient proteoform fractionation from complex biological samples using passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry (PEPPI-MS), a rapid method for extraction of intact proteins separated by SDS–PAGE. PEPPI-MS allows recovery of proteins below 100 kDa separated by SDS–PAGE in solution with a median efficiency of 68% within 10 min and, unlike conventional electroelution methods, requires no special equipment, contributing to a remarkably economical implementation. The entire protocol from electrophoresis to protein purification can be performed in &lt;5 h. By combining the resulting PEPPI fraction with other protein-separation techniques, such as reversed-phase liquid chromatography and ion mobility techniques, multidimensional proteome separations for in-depth proteoform analysis can be easily achieved.</p>