Background: Lung cancer is a leading cause of mortality with 5-year survival less than 20%, largely a result of many cases being diagnosed late. Early detection can increase cancer survival up to 13-fold underscoring the need for effective screening. Targeted Low dose computed tomography (LDCT) has been shown to be effective but its impact to date has been limited due to slow adoption and variable uptake in high-risk populations. Breath analysis represents a non-invasive screening approach either alone or alongside LDCT. Numerous studies have investigated potential endogenous breath biomarkers of lung cancer. Many have produced promising results but to date, no validated biomarkers with clear connections to cancer metabolism have been revealed. We have explored an alternative, probe-based approach based around Exogenous Volatile Organic Compound Probes (EVOC Probes). The probes target tumour associated extracellular b-glucuronidase, a glycosidase enzyme that normally resides within lysosomes. Methods: We use a hydrophilic non cell permeable substrate probe D5-ethyl-βD-glucuronide (D5-EtGlu) that upon hydrolysis by the target enzyme releases D5-ethanol, a unique volatile reporter molecule detectable on breath. This provides a readout of tumour associated enzyme activity using breath analysis. Results: Administering D5-EtGlu to mice resulted in tumour specific release of D5-ethanol, enabling discrimination between healthy and tumour bearing animals. Increased expression of b-glucuronidase in lung cancer tissue and the tumour microenvironment was confirmed with immunohistochemistry (IHC) in clinical samples. A phase 1a clinical trial administered D5-EtGlu to healthy individuals in a single ascending dose study to establish safety and background D5-ethanol levels in healthy individuals. This resulted in no adverse events and low/no D5-ethanol signal verifying the inaccessibility of D5-EtGlu to intracellular b-glucuronidase. The next stage, currently ongoing, is a proof of mechanism in humans. D5-EtGlu is administered intravenously to confirmed lung cancer patients followed by breath analysis. D5-ethanol breath levels will be compared to cancer free individuals receiving the same dose of D5-EtGlu. Conclusions: Non-invasive breath testing has great potential to contribute to diagnosis for lung cancer including a potential role in screening. Our current work is evaluating the use of an administered probe to stimulate tumour-specific enzyme activity and produce a marker detectable on breath. Continued success could result in a sensitive and highly specific method for lung cancer early detection.