This work presents an in-depth study on the nature of the faecal pigment-zinc(II) complexes using spectroscopic, thermodynamic and computational studies towards a molecular level understanding regarding its structure, complexation and stability. The analysis of faecal pigments (FPs), specifically urobilin (UB) and stercobilin (SB), is often carried out using Schlesinger's test, that uses the zinc complexation induced enhancement of FPs fluorescence. It was observed that in addition to the expected 1:1 stoichiometry, FP-Zn(II) complex with 1:2 stoichiometry also form. This was confirmed by the ESI-MS mass spectral fragmentation patterns. A rigorous benchmarking process towards using optimized basis set and functionals for molecular orbital TD-DFT calculations on UB-Zn(II) complex suggested the use of B3LYP hybrid functional and LANL2DZ basis set as the optimized combination. Molecular geometries and transition energies predicted from MO calculations corroborated well with FTIR and mass spectrometric results. FP-Zn(II) complexation equilibria in aqueous media involve disaggregation of higher FPs followed by metal-ligand complexation. The binding constant values of FP-Zn(II) complexes were estimated to be 101 M−1 & 22 M−1 for 1:1 and 1:2 stoichiometries of SB-Zn(II) complexes and 105 M−1 & 11 M−1 for 1:1 and 1:2 stoichiometries of UB-Zn(II) complexes in aqueous media. For the 1:1 complexes, the enthalpy (∆H0) and entropy (∆S0) values for SB-Zn(II) are 65 k J mol−1 and 269 J K−1 mol−1, respectively. Similarly, enthalpy (∆H0) and entropy (∆S0) for UB-Zn(II) complexes are 69 kJ mol−1 and 256 J K−1 mol−1, respectively. This understanding of the structure and thermodynamics of FP-Zn(II) complex is expected to aid in developing aqueous phase analytical strategies towards the fluorescence-based analysis of FPs, an important component of water quality analysis. © 2021