Determination of the 4-hydroxy-l-proline (hydroxyproline) concentration may provide useful information for the diagnosis and prognosis of diseases caused by disorders of collagen metabolism. The objective of the present study was to apply liquid chromatography-mass spectrometry (LC-MS) to assess the hydroxyproline concentration. The hydroxyproline concentration in lung and liver tissues measured by LC-MS was compared with values obtained by a colorimetric method, as well as a fluorescence method using high-performance liquid chromatography (HPLC) from previous studies by our group. The determination of the hydroxyproline concentration by LC-MS was improved as compared with that using the colorimetric and HPLC methods, due to its simplicity, high sensitivity (pg level) and short separation time. These results suggested that utilizing the LC-MS method for measuring the hydroxyproline concentration would be advantageous for the diagnosis of diseases associated with abnormalities of collagen metabolism.