We have explored a general approach for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry. We engineered a gene to express green fluorescent protein (GFP) with a synthetic fusion protein (GAB-GFP) in E. coli to function as a spectroscopic standard for the quantification of an analogous stable isotope labeled, non-fluorescent fusion protein (GAB*), and for the quantification and stoichiometric analysis of purified transducin, a heterotrimeric G-protein complex. Both GAB-GFP and GAB* contain concatenated sequences of specific proteotypic peptides that derive from the a, ß and  protein subunits of transducin and that are each flanked by spacer regions that maintain the native proteolytic properties for these peptide fragments. Spectroscopic quantification of GAB-GFP provided a molar scale for mass spectrometric ratios from tryptic peptides of GAB*, and defined molar responses for mass spectrometric signal intensities from a purified transducin complex. The stoichiometry of transducin subunits a, ß and  was measured to be 1:1.1:1.15 over a 5-fold range of labeled internal standard with an RSD of 9%. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic or antibody-based technologies.