Clinical blood biomarkers provide important diagnostic information and are generally secreted, cytoplasmic, membrane and other soluble proteins. However, in the mass spectrometry-based proteomics-based discovery phase, the whole tissue is often lysed and analyzed, increasing protein complexity and thus limiting the discovery of those soluble biomarkers. Here we use a mild extraction method that utilizes low salt buffer and 1% Triton X-100 to extract proteins from the tissue sample followed by the proteome analysis. This mild condition extracts membrane and cytoplasmic proteins efficiently while spares nuclear proteins largely from the mouse brain tissue as demonstrated by Western blot. The subsequent proteomic analysis shows significant enrichment of secreted and cytoplasmic proteins in the low salt and Triton extracts. Expanding application of this method to other 16 mouse tissue samples has yielded similar results. Further in-depth proteomic analysis of an Alzheimer mouse brain tissue mild extract has identified more than 12,000 proteins with significant enrichment in the soluble proteins and decent depth of serum proteins. Overall, this mild tissue extraction demonstrates to be an efficient approach for proteomic discovery of circulating biomarkers.