Proteome analysis is typically performed by direct integration of chromatography and mass spectrometry, allowing unattended and standardized analysis of multiple samples. In contrast, sample preparation prior to LC-MS is still largely a manual process, constituting a major source of variability in sample handling and, ultimately, in proteomic data. Here, we present how we established a generic, automated workflow for proteomic sample preparation by combining AFA-based ultrasonication and single-pot solid-phase-enhanced sample preparation (SP3), enabling routine and standardized proteome analysis with minimal hands-on time. Sonication is used for protein extraction from virtually any sample type, including cells, fresh tissue and FFPE tissues in a 96-well format, followed by protein clean-up and digestion by SP3 performed on a Bravo liquid handling platform. Sensitivity and reproducibility of the approach will be shown from low-input experiments starting from 100-1000 cells, from fresh tissues of various organs, and from FFPE tissue sections of cancer specimens. Furthermore, we used this pipeline to analyze a cohort of 96 ependymoma brain tumor samples, and integrated the obtained proteomic data with other omics layers for disease sub-classification. In conclusion, AFA-based ultrasonication combined with autoSP3 is a robust platform for standardized and parallel processing of a variety of tissue types for low-input proteomics, serving a wide range of clinical and non-clinical proteomic applications.