While yeast is one of the most studied organisms, its intricate biology remains to be fully mapped and understood. This is especially the case when it comes to capture rapid, in vivo fluctuations of metabolite levels. Secondary electrospray ionization-high resolution mass spectrometry SESI-HRMS is introduced here as a sensitive and noninvasive analytical technique for online monitoring of microbial metabolic activity. The power of this technique is exemplarily shown for baker’s yeast fermentation, for which the time-resolved abundance of about 300 metabolites is demonstrated. The results suggest that a large number of metabolites produced by yeast from glucose neither are reported in the literature nor are their biochemical origins deciphered. With the technique demonstrated here, researchers interested in distant disciplines such as yeast physiology and food quality will gain new insights into the biochemical capability of this simple eukaryote.