Biochemical evaluation of lung collagen content is considered one of the gold standard endpoints in the preclinical assessment of novel therapeutic agents for IPF. For the evaluation of collagen levels in lung tissues, several methodologies have been described, including histological analysis, gene expression, HPLC and colorimetric assays. However, some of these techniques may not always reflect the lung collagen accumulation and fibrosis in in vivo models. For instance, some assays allow only the detection newly formed collagen, not taking into account the insoluble collagen of the scar tissue fraction1. We aimed to validate a robust and reproducible hydroxyproline technique that allows accurate assessment of collagen deposition. For this purpose, we analysed sample stability, intra-assay precision and linearity of samples used in the hydroxyproline assay. Finally, we correlated the validated biochemical readout with histological analysis.