Non-invasive molecular imaging techniques are accruing more interest in the last decades. Several radiolabelling elements have been FDA-approved and are currently used to characterize tumors. In this study, the DNA intercalating agent doxorubicin was radiolabelled with ¹²⁵I. Several parameters for the radiolabelling reaction were investigated and opti- mized. A maximum yield of 94 ± 0.3% was reached after reacting 20 lg of doxorubicin with 200 μg Chloramine-T at pH 5 for 30 min. The in vivo stability of 125I-doxorubicin is validated by the low propensity for thyroid uptake in mice. The preclinical T/NT ratio was approximately 6.4 at 30 min. Docking and molecular dynamics confirmed that the radiolabelling of doxorubicin did not affect (or slightly improved its binding to DNA). Overall, ¹²⁵I-doxorubicin was demonstrated to be a promising non-invasive probe for solid tumor imaging.