IPTG (Isopropyl-β-d-1-thiolgalactopyranoside) is a gratuitous inducer commonly used for the overexpression of heterologous recombinant proteins in Escherichia coli. A reliable method has been developed for the determination of IPTG in E.coli fed-batch fermentation samples with a minimal sample treatment. Analysis was performed in single ion monitoring positive mode using ESI source. The extracted ion was 261 m/z and the retention time of IPTG was 12.4 min with a total run time in samples of 30 min. The flow was directed to mass spectrometer 11 min after the start of the run and diverted from mass spectrometer after 14.5 min in order to avoid interference of salts and other metabolites. The assay was validated for medium and intracellular matrices and linear calibration curves of 3 orders of magnitude were obtained (R2 ≥ 0.99). Quality control samples were analyzed and showed precision and accuracy within the limits according to FDA Guidelines for analytical method validation. Recovery for both matrices was between 95.8 and 113.5%. The limit of detection (LOD) was set at 0.02 μM being the 0.1% of the lowest IPTG concentration used for induction of recombinant protein overexpression. The developed procedure has been applied to determine the IPTG distribution profiles in medium and intracellular samples in high cell density induced cultures for the production of the recombinant protein rhamnulose-1-phosphate aldolase (RhuA).