Monitoring the biological fate of medicaments within the environments of cancer cells is an important challenge which is nowadays the object of intensive studies. In this regard, rhodamine-based supramolecular systems are one of the most suitable probes used in drug delivery thanks to their high emission quantum yield and sensitivity to the environment which helps to track the medicament in real time. In this work, we used steady-state and time-resolved spectroscopy techniques to investigate the dynamics of the anticancer drug, topotecan (TPT), in water (pH ~6.2) in the presence of a rhodamine-labeled methylated β-cyclodextrin (RB-RM-βCD). A stable complex of 1:1 stoichiometry is formed with a Keqvalue of ~4 × 104M−1at room temperature. The fluorescence signal of the caged TPT is reduced due to: (1) the CD confinement effect; and (2) a Förster resonance energy transfer (FRET) process from the trapped drug to the RB-RM-βCD occurring in ~43 ps with 40% efficiency. These findings provide additional knowledge about the spectroscopic and photodynamic interactions between drugs and fluorescent functionalized CDs, and may lead to the design of new fluorescent CD-based host–guest nanosystems with efficient FRET to be used in bioimaging for drug delivery monitoring.