Purpose: Keratoconus (KC) is a multifactorial, ectatic corneal disease. Metabolic changes in the corneal stroma with alterations in collagen fibril stability, oxidative stress, and urea cycle, have previously been reported as key players in KC pathobiology. Recently, corneal collagen cross-linking (CXL) has been introduced as a treatment that can address the progressive nature of KC. While the treatment has been successful in the early days, it is not without clinical ramifications. In this study, we investigated the alterations in KC metabolic profiles due to CXL. Methods: Primary human corneal fibroblasts (HCFs) from healthy donors and human KC fibroblasts (HKCs) from KC donor patients were plated on transwell polycarbonate membranes and stimulated by a stable vitamin C. At 4 weeks, riboflavin was added to the cultures followed by UVA irradiation (365 nm). Using mass spectrometry, we measured the major differences in metabolites in HKCs compared to HCFs pre- and post CXL. Result: The analysis of 276 metabolites in HCFs and HKCs revealed that the most affected metabolites due to CXL were glutathione disulfide, ascorbic acid, proline, and lysine. A significant decrease in the pro-inflammatory biomarkers (myo-inositol and histidine) was also observed. Furthermore, a significant downregulation of many amino acids, lactate levels, and other water-soluble metabolites was noted in HKCs following CXL. Conclusion: CXL is a KC treatment available to patients within certain criteria. Surprisingly, the cellular and molecular mechanisms are considerably understudied limiting our ability for more precise and targeted CXL treatments. In this study, for the first time, we report the effects of CXL on KC metabolism.