In this study, the suitability of amide proton transfer (APT) imaging as a biomarker for the characterization of high intensity focused ultrasound (HIFU)-treated tumor tissue was assessed.APT imaging was performed on tumor-bearing mice before (n = 15), directly after (n = 15) and at 3 days (n = 8) after HIFU treatment. A control group (n = 7) of nontreated animals was scanned at the same time points. Histogram analysis of the tumor APT-weighted signal distributions was performed to assess HIFU-induced changes in the tumor APT contrast.Distinct regions of decreased APT-weighted signal were observed at both time points after HIFU treatment. Analysis of the tumor APT-weighted signal distribution showed a pronounced shift toward lower APT-weighted signal values after HIFU treatment. A significantly increased fraction of pixels with an APT-weighted signal value between -10 and -2% was observed both directly (0.37 ± 0.16) and at 3 days (0.49 ± 0.16) after HIFU treatment as compared to baseline (0.22 ± 0.16).The presented results show that APT imaging is sensitive to HIFU-induced changes in tumor tissue and may thus serve as a new biomarker for monitoring the response of tumor tissue to HIFU treatment.