Sertraline (SRT) is a widely used antidepressant whose poor solubility in water limits its oral applicability. Thus, the aim of this work was the evaluation of a multi-equilibrium system based on β-cyclodextrin (βCD) and SRT. The inclusion compounds (ICs) were investigated by infrared spectroscopy, isothermal titration calorimetry (ITC) and 1H and 2D ROESY nuclear magnetic resonance experiments. SRT solubility was predicted in vitro in water and biomimetic fluids. The SRT in presence of βCD at 1:1 and 1:2 molar ratios was more soluble than free SRT in all biomimetics media investigated. The FTIR-HATR showed that βCD νC–O–C stretching band was reduced in presence of SRT, suggesting the interactions between them. Additionally, titration process and Job's plot provided information on the ICs stoichiometry and evidenced the multi-equilibrium coexistence in aqueous solution. According to the ITC, SRT:βCD interaction process was spontaneous and exothermic with a high affinity binding constant (K = 14,726 M−1). Additionally, the stoichiometry coefficient (n) was 1.63, which was comparable to that found by FITR-HATR. The 1H and 2D ROESY verified multiple SRT sites included into the host cavity. Theoretical calculations depicted the relative energy of different proposed ICs structures, in which the 1:2 IC was the most stable.