Next generation clinical mass spectrometry that relies on SWATH data acquisition is transforming translational research. High resolution mass spectrometry instruments are so effective that the sample preparation has become the key limiting factor for consistent and reproducible large scale protein quantitation. Here, we have developed a high-throughput and fully automated sample preparation pipeline that integrates the key steps in SWATH-based proteomic workflow, including digestion, desalting and fractionation to build comprehensive pan-species spectral repositories and collect quantitative profiles for 96 samples. The pipeline utilizes a liquid handling robot and 96-well filter plates for efficient FASP-based protein digestion. Desalting and fractionation are achieved within the same system using vacuum-aided STAGE tips filled with SCX membrane without compromising reproducibility. We leveraged the improved throughput to create a compendium of cattle proteins from 24 clinically relevant tissues and body fluids and demonstrated uniform and effective digestion performance across all tissue types that allowed us to reproducibly quantify nearly 6,000 proteins using SWATH-MS. This end-to-end automated proteomic sample preparation overcomes technical barriers, minimizes the cost and enables accurate quantitative proteomic analysis across any biological system.