PEGPH20 has been shown to decompress blood vessels and improve tumour drug delivery. Given the sensitivity of R2* to paramagnetic deoxyhaemoglobin and reflection of RBC content in tumour vasculature, we hypothesised that IS-MRI may provide a useful biomarker of PEGPH20 response. We performed IS-MRI before and after PEGPH20 treatment in 4T1 HAS3 and MDA-MB-231 LM2-4 orthotopic breast tumours. R2* significantly increased following PEGPH20 treatment in 4T1 HAS3 tumours, however, R2* was unable to detect a similar response to PEGPH20 in the relatively hypovascular MDA-MB-231 LM2-4 tumours. Another vascular biomarker may provide greater sensitivity and detect PEGPH20 response in hypovascular tumours.