Here we describe a liquid chromatography coupled inline to mass spectrometry (LC-MS) method for the quantification of fluorinated nucleosides in RNA and DNA. 5-fluorocytosine (5FC) is a nucleoside analogue that acts as an antimetabolite following intracellular deamination to 5-fluorouracil. It is a frequently used antifungal medication. There is interest in using 5FC to treat drug-resistant Pseudomonas aeruginosa as these chronic infections present a risk to cystic fibrosis patients. 5FC can be metabolised in a number of ways and its mode of action in P. aeruginosa is not yet known. A microflow LC-MS method was established using a Luna Omega polar C18 column (1.7 µm, 1 mm ID x 150 mm) to separate hydrolysed and dephosphorylated nucleosides from the RNA and DNA of P. aeruginosa that had been grown in the presence of sub-lethal amounts of 5FC. The method targeted uridine, thymidine, 5-fluorouidine (5FU), and 2-deoxy-5-fluorouridine (d5FU) for quantification on a 5600 Triple TOF mass spectrometer (AB Sciex). The assay exhibited good reproducibility and sensitivity, with lower limits of quantification ranging from 10 – 160 fmols. The accuracy was better than 15% across the entire range of quantification for each analyte. The results showed that 5-fluorocytosine treatment led to 4.2- 7.2% of uridines in RNA from P. aeruginosa being fluorinated. 5FU and d5FU were not detected in DNA from 5FC treated P. aeruginosa. Therefore, targeted LC-MS was a powerful tool for detection and quantification of metabolites of 5FC in RNA. The results demonstrate that the antimicrobial effects of 5FC on P. aeruginosa are due at least in part to incorporation of 5FC derivatives into bacterial RNA.