A bioanalytical method for the quantitative determination of budesonide and fluticasone in human sputum was developed. Sputolysin ® Reagent was added to the sputum samples. After incubation (37°C; 60-70min under shaking) and automated solid phase extraction the extracts were analysed using LC-MS/MS. Budesonide and fluticasone showed good linearity (r>0.99) over the range 0.1-100nM in the first and second validation batch, and over the range 0.25-10,000nM in the third and fourth validation batch. The lower limit of quantification (LLOQ) achieved was 5nM for budesonide and fluticasone in 100μL human sputum. Intra-run and inter-run RSD for four quality control levels (5-100nM) were within 6.9% (budesonide) and 8.0% (fluticasone). The accuracy ranged from -11.4% to -1.6% (budesonide), and from -11.8% to 0.4% (fluticasone). The validated method was applied to clinical sputum samples from COPD patients. © 2011 Elsevier B.V.