Efficient homogenization of small amount of samples, such as biopsy-level tissues, and reproducible protein extraction from these samples, remain outstanding technical challenges for protein analyses in clinical research, particularly in the coming era of precision medicine. We have recently presented an integrated method based on pressure cycling technology and pressure-resistant MicroTubes to efficiently perform tissue homogenization and protein extraction with minimal manual operation, assisted by the Barocycler. In order to further enhance the efficiency of this method, here we present an improved extraction method which utilizes the PCT μPestle, a disposable mechanical tissue homogenizer that is incorporated directly into the MicroTube sample container. The container walls collapse around the insert under repeated pulses of high hydrostatic pressure, resulting in a homogenizer-like effect. Pressure cycling also acts directly on the sample, resulting in more efficient tissue lysis. We optimized the pressure cycling conditions for μPestle, and further evaluated the performance of the optimized μPestle method using our conventional MicroCap method as a benchmark. Comparison of extraction yields from the two methods using mouse liver, heart, and brain tissues of different amounts (ranging from 0.5 mg to 13 mg) led to the same conclusion, that μPestle extracted significantly more proteins than the MicroCap method. We further integrated the μPestle method into the PCT-based protein digestion and SWATH-MS analysis workflow [1], and found the optimized μPestle method produced 20-40% more peptides for various tissues and identified more biologically informative proteins after SWATH analysis when compared to the MicroCap method. In conclusion, we have developed a hands-free method for efficient and reproducible tissue homogenization and protein extraction, and integrated it into the PCT-SWATH workflow which rapidly converts small amount of samples into a digital map representing the MS-measurable proteome.