Type I collagen, which consists of two α1 and one α2 chains, is an abundant extracellular matrix ( ECM ) protein. Changes in type I collagen are often associated with various diseases including fibrosis, osteogenesis imperfecta ( OI ), and Ehlers-Danlos syndrome ( EDS ). In the present study, we developed a method for quantification of type I collagen α1 ( COL1A1 ) in a culture medium of LX-2 human hepatic stellate cells by using nano-liquid chromatography tandem mass spectrometry ( nano-LC/MS/MS ). After selecting a specific peptide of COL1A1, unlabeled and stable isotope-labeled peptides were used for quantitative analysis of COL1A1 by nano-LC/MS/MS. The concentration of secreted COL1A1 in the LX-2 cell culture medium was 38.78 ng/mL. The results indicate that this method is useful for quantifying COL1A1 in a cell culture medium.