In industrial hygiene, biomarkers maintain their promise to reveal the true extent of occupational exposure. The environmental limit values proposed by industrial hygienist associations are more than biological, health-based values indicating professional hazards to humans. High-throughput screening of samples is therefore the strategy of choice to detect occupational exposure biomarkers, yet it requires user-friendly apparatus that give relatively prompt results while ensuring high degrees of selectivity, precision, accuracy and automation, particularly in preparation processes. In light of the above, this contribution describes a novel gas chromatography/triple quadrupole mass spectrometry/positive chemical ionisation approach for determining urinary α-fluoro-β-alanine, a metabolite of 5-fluorouracil, the most widely employed antineoplastic drug. In this new procedure chromatography’s sensitivity is combined with the user-friendliness of alkyl chloroformate/trialkyloxonium on-sample derivatizations followed by solid-phase microextraction sampling, to which is added the quantitative accuracy afforded using a specific isotope-labelled internal standard. The quantification limit for α-fluoro-β-alanine was 25.4 µg/L. Intra- (3.8%) and inter-session (4.5%) repeatability was also evaluated. This method serves to identify suitable risk-control strategies for occupational hygiene conservation programs. © 2017, Malaysian Society of Analytical Sciences. All rights reserved.