AIM: Airway mucins in sputum are promising respiratory disease biomarkers, despite posing substantial analytical challenges due to their physicochemical properties and rare and heterogenous nature of the matrix. We aimed to identify a suitable sputum collection and processing method, and qualify a bioanalytical method for MUC5AC and MUC5B quantification in clinical samples. METHOD: Mucins were quantified in induced and spontaneous sputum collected from the same COPD patients, following various sample processing procedures. LC-MS/MS method used truncated recombinant mucins as surrogate analytes in surrogate matrix. RESULTS: Frozen spontaneous sputum was found to be a suitable and convenient matrix for mucin quantification and fit-for-purpose method qualification was performed. CONCLUSION: Our methodology provides accurate and reliable MUC5AC and MUC5B quantification and facilitates multi-site clinical sputum collection.