The present study reports a meticulous characterization of the interaction of a potent anti-cancer drug, berberine chloride (BR) with a series of bile salt aggregates having varying hydrophobicity (sodium deoxycholate (NaDC), sodium cholate (NaC), and sodium taurocholate (NaTC)). The absorption spectrum of BR reveals a complex profile comprised of four distinct peaks. Analysis of the modulations of the absorption spectral properties of BR following interaction with the bile salts raising deeper questions unveils a critical insight into the mode of interaction of the cationic drug (BR) with the bile salts; a greater degree of perturbation of the microenvironment of the isoquinolinic part of BR compared to the benzenoid part. The remarkable modulation of the fluorescence profile of BR with added bile salts provides a sensitive indicator for monitoring the interaction scenario. However, an intriguing observation in this context reveals differential fluorescence behavior of BR in various bile salt aggregates, that is, similar observations in NaDC and NaC (which are legitimately interpreted according to the 'two-step association model') in comparison to NaTC. Such contrasting behavior of BR in NaTC aggregates has been rationalized on the basis of the possibility of formation of dye aggregate facilitated because of the proximity of the cationic drug molecules to the anionic headgroup of NaTC bile salt. Surprisingly, our spectroscopic results evidence for binding location of the drug at the interfacial region in all the bile salt aggregates. To this end, the time-resolved fluorescence decay behavior of the drug within various bile salt aggregates has been meticulously studied. The fluorescence decay results are found to be highly sensitive to the structure and size of the bile salt aggregates eventually leading to characterization of the interaction of the drug with the bile salts in excellent corroboration with the steady-state data. Furthermore, the time resolved fluorescence anisotropy decay measurements yielded insight into the modulation of rotational dynamical behavior of the drug within the bile salt aggregates. (C) 2017 Elsevier Inc. All rights reserved.