The natural ability of Pseudomonas taiwanensis VLB120 to use xylose as sole carbon and energy source offers a high potential for sustainable industrial biotechnology. In general, three xylose assimilation routes are reported for bacteria. To elaborate the metabolic capacity of P. taiwanensis VLB120 and to identify potential targets for metabolic engineering, an in silico/in vivo experiment was designed, allowing for discrimination between these pathways. Kinetics of glucose and xylose degradation in P. taiwanensis VLB120 was determined and the underlying stoichiometry was investigated by genome-based metabolic modelling and tracer studies using stable isotope labelling. Additionally, reverse transcription quantitative polymerase chain reaction experiments have been performed to link physiology to the genomic inventory. Based on in silico experiments, a labelling strategy was developed, ensuring a measurable and unique (13) C-labelling distribution in proteinogenic amino acids for every possible distribution between the different xylose metabolization routes. A comparison with in vivo results allows the conclusion that xylose is metabolized by P. taiwanensis VLB120 via the Weimberg pathway. Transcriptomic and physiological studies point to the biotransformation of xylose to xylonate by glucose dehydrogenase. The kinetics of this enzyme is also responsible for the preference of glucose as carbon source by cells growing in the presence of glucose and xylose.