Ultraviolet A (UVA)-irradiated 4′-hydroxymethyl-4,5′,8-trimethyl psoralen (HMT) in the presence of a poly-dT17 and dA7TTA8 oligonucleotides produces HMT–dT17 and HMT–dA7TTA8 adducts in aqueous solution. In this article, we determine whether these HMT–dT17 and HMT–dA7TTA8 adducts can be detected with a molecular beacon (MB) probe. We measure the degree of damage in dT17 and dA7TTA8 solutions containing UVA-activated HMT via monitoring the decrease in MB fluorescence. Photoproduct formation is confirmed by MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-fight mass spectrometry measurements) and absorption spectroscopy. The MB fluorescence decreases upon UVA irradiation in the presence of HMT with a single-exponential time constants of 114.2 ± 6.5 min for HMT–dT17 adducts and 677.8 ± 181.8 min for HMT–dA7TTA8 adducts. Our results show that fluorescent MB probes are a selective, robust and accurate tool for detecting UVA-activated HMT-induced DNA damage.