The development of rapid analytical strategies plays a vital role for the research, discovery and confirmation of analytes that can be sensitive biomarkers. Within this line of work, a method based on headspace-gas chromatography-mass spectrometry has been developed and validated for the determination of possible biomarkers (dimethyl disulfide, toluene, 1,4-xylene, 2-octanone and 2,6-dimethyl-7-octen-2-ol) in human urine samples. The matrix effect was analyzed and the optimal analytical conditions were determined. The limits of detection ranged from 0.01 to 0.48 μg L⁻¹, whereas the limits of quantification ranged from 0.05 to 1.43 μg L⁻¹. The repeatability and reproducibility of the proposed method were below 9.5% and 14.2%, respectively. Five urine samples from healthy subjects, spiked with the possible biomarkers, were used to validate the proposed methodology. The quantification of these compounds in three urine samples from healthy subjects was performed with the one-point standard additions protocol.