An analytical approach employing headspace sorptive extraction coupled with gas chromatography-mass spectrometry (HSSE-GC-MS) has been successfully developed for the determination of apocarotenoid volatiles arising from the enzymatic activity of carotenoid cleavage enzymes (CCDs) in Escherichia coli. The GjCCD4a enzyme derived from gardenia, known for its cleavage specificity at 7,8 and 7',8' double bonds across diverse carotenoid substrates, was utilized as a reference enzyme, using β-carotene as the substrate for the enzymatic activity assays. Optimal headspace conditions for analysis were established following a 5 hours induction period of the recombinant GjCCD4a protein within E. coli cells, engineered to produce β-carotene. The analytical method demonstrated linearity, with correlation coefficient (R2 > 0.95) in calibration, while achieving detection and quantification limits conducive to the accurate determination of β-cyclocitral. Notably, this methodological framework significantly reduced both the handling complexity and sample processing time in comparison to conventional liquid chromatography methods employed for the detection of cleavage products and determination of CCD activities. The proposed HSSE-GC-MS approach not only enhances the efficiency of apocarotenoid analysis but also provides a sensitive means for unraveling the intricate enzymatic processes associated with CCD-mediated carotenoid cleavage in a bacterial model system.