Qualitative and quantitative changes in collagens are linked to various diseases. Monitoring of collagens may provide new insights into the assessment and diagnosis of collagen-related diseases. In this study, we established a method that enables simultaneous quantification of the α chains of types I, III, and V collagens, namely COL1A1, COL3A1, and COL5A1, in a culture medium of LX-2 human hepatic stellate cells using nano-liquid chromatography tandem mass spectrometry (nano-LC/MS/MS). Specifically, simultaneous quantification of multiple collagen α chains could be accomplished by using their specific target peptides. Compared with conventional enzyme-linked immunosorbent assay (ELISA), this method is more sensitive and efficient for simultaneous quantification of various collagens. These data suggested that our newly developed quantification method is valuable and could be used for the diagnosis of collagen-related diseases.