The encapsulation of testosterone and it aliphatic dimer (alip) and aromatic dimer (arom) with milk β-lactoglobulin (β-LG) was studied in aqueous solution at pH 7.4. Multiple spectroscopic methods, transmission electron microscopy (TEM) and molecular modeling were used to characterize testosterone-β-LG binding and protein aggregation process. Spectroscopic analysis showed that steroids bind β-LG via hydrophobic and H-bonding interactions with overall binding constants K test--β-LG =5.6 (±0.6)×10 4 M −1 ,K test-dimer alip-β-LG =4.8 (±0.5)×10 3 M −1 and K test-dimer-arom-β-LG =2.9 (±0.4)×10 4 M −1 . The binding affinity was testosterone>testosterone dimer-aromatic>testosterone dimer-aliphatic. Transmission electron microscopy showed major changes in protein morphology as testosterone–protein complexation occurred with increase in the diameter of the protein aggregate indicating encapsulation of steroids by β-LG. Modeling showed the presence of H-bonding stabilized testosterone-β-LG complexes with the free binding energy of −9.82Kcal/mol indicating that the interaction process is spontaneous at room temperature.