Surfactants are surface-active agents applied in many aspects and products in our daily lives. Biosurfactants are fully bio-based surfactants and a new type of innovative microbial biosurfactants, bola sophorosides, has recently been developed at InBio.be. However, the low productivities of the biosynthesis do not allow the process to be economically viable. This will be tackled in this project by performing bioprocess development in fed-batch and continuous setups. Typically such bioprocess development is combined with targeted and off-line analyses of substrate and bioproduct. However, these analyses do not provide any information about unknown underlying biochemical ‘marker’ pathways/metabolites influencing biosurfactant production and the off-line, delayed follow up often results in actions not taken timely. Therefore a metabolomics approach will be set up in parallel to the bioprocess development. The metabolic fingerprint will be monitored using untargeted rapid evaporative mass spectrometry and correlated to the bioprocess efficiency. Furthermore, an at-line integration of the bioprocess with the metabolomics platform will be investigated. This metabolomics approach, reflecting the complex interplay between microbial kinetics and dynamic environmental changes in bioreactors, will speed up bioprocess development. Moreover, a successful at-line metabolomics approach would be a very powerful tool in monitoring (industrial) fermentations.