Background: Desmosine/isodesmosine (DES/IDS) is a promising biomarker for estimating activity of elastin degradation. Results/methodology: A stable isotope dilution LC–MS/MS method for measuring serum/plasma DES/IDS was developed and validated. The reportable range of this assay was 0.1–160 ng/ml. Serum/plasma DES/IDS level was stable at room temperature or 4°C for 20 h, and for three freeze–thaw cycles. Interferences from endogenous compounds and ion suppression/enhancing effect were also evaluated. Our results suggest the absolute necessity of using an IS in the measurement. We found that serum/plasma DES/IDS levels from patients with chronic obstructive pulmonary disease and cystic fibrosis were significantly higher compared with healthy smokers. Conclusion: These results demonstrate that the LC–MS/MS method provides sensitive, reproducible and accurate quantification of total serum/plasma DES/IDS.