Sufentanil (SUF) is a synthetic analgesic opioid widely used for the management of acute and chronic pain. This drug was complexed with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and the physicochemical characterization, in vitro/ex vivo toxicity assays, and pharmacological evaluation were performed. Differential scanning calorimetry, Fourier transform infrared spectroscopy (FTIR) analysis, and X-ray powder diffraction showed the formation and the morphology of the complex. Nuclear magnetic resonance afforded data regarding inclusion complex stoichiometry (1:1) with an association binding constant (Ka) value of 515.2 ± 1.2M-1 between SUF and HP-β-CD. Complexation with HP-β-CD protected SUF from light exposure and increased its photostability. Release kinetics revealed a decrease in SUF release rate (Krel = 7.05 ± 0.52 and 5.61 ± 0.39min-1/2 for SUF-HP-β-CD and SUF, respectively) and reduced hemolytic or myotoxic effects after complexation. 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