L-Hydroxyproline (L-Hyp) is an important biomarker for idiopathic pulmonary fibrosis (IPF). The quantitative methods based on high-performance liquid chromatography coupled with fluorescence detection after pre-column derivatization typically requires complicated derivatization conditions and obtains unstable derivatives. Here, a novel derivatization reagent, 9-acetylimidazol-carbazole, was synthesized for the first time to efficiently and rapidly label the amino groups of L-Hyp. The high-performance liquid chromatography method with pre-column derivatization was performed on an Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm). The product was measured using fluorescence detection at excitation and emission wavelengths of 232 and 370 nm, respectively. The method was validated in specificity, linearity, limit of detection (66.7 fmol), limit of quantification (333.3 fmol), intra-day precision (0.75%), inter-day precision (3.82%), stability (3.15%), and recovery (90.7–109.4%). The validated method was successfully applied to the determination of L-Hyp in the lung tissues of healthy and IPF rats. The results showed that the concentration of L-Hyp (3.64 mg/g) in the IPF model was significantly higher than the concentration (2.33 mg/g) in the healthy control group with P < 0.01. This is a new method for the determination of L-Hyp and can be used for other amino acid-related studies in the future.