Different methodologies for collagen quantification have been described in the past. Introduction of mass spectrometry combined with high-performance liquid chromatography (HPLC) is a high-resolution tool, which has generated novel applications in biomedical research. In this study, HPLC coupled to electrospray ionization (ESI) tandem mass spectrometry (HPLC-ESI-MS/MS) was used to characterize tissue samples from AVFs done in rats. These findings helped create a protocol for identifying and quantifying components of immature and mature collagen crosslink moieties. Two different internal standards were used: epinephrine and pyridoxine. Quantification curves were drawn by means of these standards. The goal of the experiment was to achieve accurate quantification with the minimum amount of sample. Time and cost of experiment were considerably minimized. Up to date, this method has not been tested for crosslinking quantification.