Ln(III) complexes of macrocyclic ligand H4dota and its derivatives are utilized in many areas of medicinal chemistry. Crucial factor for MRI and luminescence probes is determination of the number of the water molecules coordinated to Ln(III) complex, then its thermodynamic stability and kinetic inertness of the complexes. Time-resolved luminescence spectroscopy of Eu(III) ion and its complexes with macrocyclic DOTA-like ligands as model Ln(III) complexes in solutions for measurement of luminescence decay is used for the determination of the number of water molecules in the first coordination sphere. This measurement can be used for estimation of the number of coordinated water molecules in the course of Eu(III) complexation by ligands or Eu(III) complex dissociation in order to propose its reaction mechanism. The achieved results can be utilized for optimization of ligand structure for Ln(III) fast complexation/slow decomplexation.