Biofilms are single- or multi-species communities of bacteria that are enclosed in an extracellular matrix. These cells generally exhibit phenotypes that are significantly different from those of planktonic cells, yet detailed elucidation of the causality and the exact nature of this metabolic shift remains challenging. Considering the strong correlation of biofilms with pathogenicity and disease in the clinic, as well as the veritable economic impact of biofilms in other areas, a methodology for in-vivo monitoring of biofilm development is necessary. Here, we present high-resolution mass spectrometry fingerprinting as a rapid, sensitive, and generic technique for online, non-invasive monitoring of developing biofilms. The opportunistic pathogen Pseudomonas aeruginosa is used as a model system, and it is demonstrated that strain- and time-dependent changes in biofilm extracellular metabolites are easily detected.