The Renin-Angiotensin-System is a peptide hormone cascade being responsible for the regulation of blood pressure and fluid balance through a sophisticated signaling network. During recent years the local Renin-Angiotensin-System (RAS) moved increasingly into the focus of research as the expression of multiple angiotensin processing enzymes had been reported for tissues. Nevertheless, scientific progress in this field is hampered due to the obvious presence of technical caveats in the analysis of the local RAS, which are indicated by a tremendous variation of tissue angiotensin levels reported in the literature. Hence, in order to assess the functions of the local RAS in different tissues, a solid and reproducible assay for the measurement of endogenous angiotensin peptide levels in tissues would be essential. We developed a novel mass-spectrometry-based method allowing the quantification of up to 10 angiotensin metabolites in tissue samples simultaneously. This novel tool was tested for applicability in murine studies by measuring tissue and plasma levels of angiotensin metabolites in wildtype and ACE2 knockout mice treated with various RAS-interfering drugs. The study provided deep insights into the systemic and tissue specific RAS revealing drug specific responses. As expected, the knockout of ACE2 resulted in increased Angiotensin II levels in hearts of knockout animals while the kidneys were only moderately affected. Enalapril treatment significantly lowered Angiotensin II levels in all investigated tissues. Surprising tissue specific effects were observed regarding other angiotensin metabolites, which underlines the importance of the comprehensive analysis of the RAS for conclusive interpretation of pharmacologic studies and allows to draw conclusions about expression levels of RAS-enzymes in the tissue of interest. Mass spectrometry based multiplex quantification of tissue angiotensin peptides (Tissue RAS-Fingerprinting) is a potent analytic tool for applications in basic science and drug development. Multiplex analysis of the tissue RAS provides new insights that might lead to the development of novel drugs and therapeutic concepts for the treatment of cardiovascular diseases in the future.