Combination therapy of erlotinib plus bevacizumab improves progression-free survival of patients with epidermal growth factor receptor-mutated (EGFR-mutated) advanced non-small-cell lung cancer (NSCLC) compared with erlotinib alone. Although improved delivery and distribution of erlotinib to tumours as a result of the normalization of microvessels by bevacizumab is thought to be one of the underlying mechanisms, there is insufficient supporting evidence. B901L cells derived from EGFR-mutated NSCLC were subcutaneously implanted into mice, and mice were treated with bevacizumab or human IgG followed by treatment with erlotinib. The distribution of erlotinib in their tumours at different times after erlotinib administration was analysed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). We also analysed the distribution of erlotinib metabolites and the distribution of erlotinib in tumours refractory to erlotinib, which were established by long-term treatment with erlotinib. We found that erlotinib was broadly diffused in the tumours from B901L-implanted xenograft mice, independently of bevacizumab treatment. We also found that erlotinib metabolites were co-localized with erlotinib and that erlotinib in erlotinib-refractory tumours was broadly distributed throughout the tumour tissue. Multivariate imaging approaches using MALDI MSI as applied in this study are of great value for pharmacokinetic studies in drug development.