Cutaneous side effects are often observed in patients treated with chemotherapeutic agents, including those treated with epidermal growth factor receptor (EGFR) inhibitors. These side effects are not fatal but often require dose reduction of chemotherapies. The mechanisms of epidermal growth factor receptor inhibition-related dermatologic toxicities are unclear, and prophylactic approaches are not well-established. To explore the mechanisms of the cutaneous side effects induced by epidermal growth factor receptor inhibition, we analyzed the metabolome using human keratinocyte cells. We first demonstrated that afatinib and lapatinib induced apoptosis in HaCaT cells. Using liquid chromatography-mass spectrometry, we detected 676 and 482 metabolites and compounds in the cells and media, respectively. We observed diverse metabolic alterations, including glycolysis, TCA metabolism, and polyamine metabolism, and also found a change in glutathione metabolites after epidermal growth factor receptor inhibition, which led to the accumulation of reactive oxygen species. Supplementation of N-acetyl cysteine partly rescued the afatinib-induced apoptosis, suggesting that reactive oxygen species are involved in the cytotoxicity of skin cells. We observed epidermal growth factor receptor inhibitor-associated comprehensive metabolic changes in human keratinocyte cells, suggesting that oxidative stress evokes cutaneous side effects induced by EGFR inhibition.