Doxorubicin (DXR) is widely used in malignancies treatment although it induces cardiotoxicity. Although DXR-induced oxidative stress by means of redox cycling and its mitochondrial localization are well documented, the contribution of mitochondrial oxidative stress and the pathways involved have not yet been elucidated conclusively. Based upon our previous reports demonstrating the relevance of monoamine oxidase MAO in cardiac diseases, this study was aimed at investigating the role of MAO in DXR-induced cardiotoxicity. Mitochondrial ROS generation was assessed in HL-1 cardiomyocyte cell line expressing HyPer (Evrogen), a probe specific for H2O2 detection. Loss of cell viability was monitored by evaluating the release of lactate dehydrogenase (LDH) in culture medium. DXR sensitizes cells to oxidative stress (10 µM H2O2) resulting in a burst of ROS formation that is completely prevented by the monoamine oxidase (MAO) inhibitor Pargyline (Parg) (100% HyPer oxidation in DXR-H2O2 vs. no HyPer oxidation in Parg-DXR-H2O2 treated cells). At the same time, MAO inhibition resulted in a 90% decrease of LDH release caused by DXR-H2O2 co-treatment. Parallel in vivo studies demonstrated that MAO inhibition increases from 20% to 60% the probability of mouse survival following DXR administration (15 mg/Kg i.p., single injection). In conclusion, our results indicate that MAO inhibition reduces cell death by preventing cell susceptibility to oxidative stress caused by DXR treatment.