Surfactants are surface-active agents applied in many aspects and products in our daily lives. Biosurfactants are a fully bio-baseda alternative to chemical surfactants and recently a new type of innovative microbial biosurfactants has been developed within InBio.be. However, the low inherent productivities of biosynthesis do not allow the process to be economically viable at scale. Productivities are to be improved by process intensification and bioprocess development across different setups. Traditionally, bioprocess development has been combined with targeted and off-line analyses of substrate and bioproduct. Such analyses are limited in scope and do not provide any information about unknown underlying biochemical ‘marker’ pathways/metabolites influencing biosurfactant production. Furthermore, off-line sample is time-consuming and a delayed follow-up of biological processes may results in actions not taken timely, negatively influencing the efficacy and cost effectiveness of any biological process. Therefore a metabolomics approach is integrated into the bioprocess development. At any time, the metabolic fingerprint of the culture broth is analysed through untargeted rapid evaporative mass spectrometry. Changes in biochemical pathways can then be correlated to the bioprocess efficiency. Such metabolomics approach, reflecting the complex interplay between microbial kinetics and dynamic environmental changes in bioreactors, may speed up bioprocess development during the development stage towards cost-effective and robust biological processes. Moreover, a successful at-line metabolomics approach is likely to be a valuable asset in improved follow-up of (industrial) fermentations, further complimenting existing methods used in industry.