The onset of the aggregation process in aqueous bile salt (BS) solutions remains a debated question. This study aimed at investigating this point through the detection of BS monomers and small aggregates using the dye Rose Bengal. Results showed that a few small BS aggregates led to a red shift of the absorption spectrum, while the BS monomer reacted with the photo-induced semi-oxidized dye and decreased its decay rate constant. The dye was dissolved in aqueous solution (10 mmol dm -3 NaOH) of sodium cholate (NaC), deoxycholate (NaDC), chenodoxycholate (NaCDC), dehydrocholate (NaDHC), glycocholate (NaGC) glycodeoxycholate (NaGDC), taurocholate (NaTC), taurodeoxycholate (NaTDC) and the zwitterionic analogue of sodium cholate called CHAPS. BS aggregates were detected at concentrations well below the concentration above which any added bile salt molecules