Quantification of metabolites present within exhaled breath is a major challenge for on-line breath analysis. It is also important for gauging the analytical performance, accuracy, reproducibility, reliability, and stability of the measuring technology. Short-chain fatty acids (SCFAs) are of high interest for nutrition and health. Their quantification enables a deep mechanistic understanding of a wide range of biological processes and metabolic pathways, while their high volatility makes them an attractive target for breath analysis. This article reports, for the first time, the development and testing of a modular, dynamic vapor generator for the qualitative and quantitative analysis of volatile SCFAs in the gaseous phase using a secondary electrospray ionization (SESI) source coupled to a high-resolution mass spectrometer. Representative compounds tested included acetic acid, propionic acid, butyric acid, pentanoic acid and hexanoic acid. Gas-phase experiments were performed both in dry and humid (95% relative humidity) conditions from ppt to low ppb concentrations. The results obtained exhibited excellent linearity within the examined concentration range, low limits of detection and quantification down to the lower ppt area. Mixture effects were also investigated and are presented.