Diagnostic development and public health surveillance require technologies that provide specific identification and absolute quantification of protein biomarkers. Beside immunologically related techniques (e.g. ELISA), mass spectrometry (MS) is gaining increasing interest due to its high sensitivity and specificity. Furthermore, MS-based analyses are extremely accurate quantitatively, provided that suitable reference standards are available. Recently, the use of chemically synthesized isotope-labeled marker peptides for MS-based absolute quantification of proteins has led to major advances. However, we show here that the use of such peptides can lead to severe biases. In this work, we present an innovative strategy (PSAQ) which uses in vitro-synthesized isotope-labeled full-length proteins as standards for absolute quantification. As those protein standards perfectly match the biochemical properties of the target proteins, they can be directly added into the samples to be analyzed, allowing a highly accurate quantification of proteins even in prefractionated complex samples. The power of our PSAQ methodology for accurate absolute quantification of biomarkers was demonstrated both on water and urine samples contaminated with Staphylococcus aureus superantigenic toxins as typical biomarkers of public health interest.