A novel, rapid and sensitive analytical method has been developed and applied to 105 sputum samples from patients with cystic fibrosis, including 5 samples from post-lung transplant patients. This new method is specifically targeted to measure β-alanyl aminopeptidase activity which is characteristic of some important Gram-negative pathogens. Of relevance to this study are Pseudomonas aeruginosa and pathogens of the Burkholderia cepacia complex both of which are commonly associated with respiratory infections as well as increased morbidity and mortality in adult cystic fibrosis patients. The analytical method involves the addition of a novel enzyme substrate (i.e. 3-amino-N-(3-fluorophenyl)propanamide) that interacts with β-alanyl aminopeptidase to generate an exogenous volatile organic compound 3-fluoroaniline (LOD 0.02 μg mL−1; LOQ 0.06 μg mL−1). 3-Fluoroaniline was determined at 20 times above its calculated limit of quantification in the sputum samples by HS-SPME-GC-MS and then the results compared with standard culture methods and bacterial identification using MALDI-TOF-MS. Detection of 3-fluoroaniline was possible after only 8 h incubation of the sputum samples with a 95% success rate; this increased to 100% at 24 h which was well within the typical routine timeframe of 48 h. To our knowledge, this is the first demonstration of detection of P. aeruginosa by use of a custom-designed substrate to liberate a detectable and unique VOC. The very high negative predictive value (100% in this study) means such an assay could be appropriate as a screening technique for patients who are not yet colonized by this pathogen.