Local anesthetics (LA), such as bupivacaine (BVC), belong to class of chemicals that reversibly block peripheral and central nerve pathways, following regional administration causing pain relief. The complexation of the S(-) enantiomer of BVC with cyclodextrins (CD) is of great interest in the development of more efficient and safe LA formulations. This study aimed to develop and to evaluate the in vitro cytotoxicity of inclusion complex with S(-)BVC and hydroxypropyl-beta-cyclodextrin (HP-beta-CD). The inclusion complex was obtained by mixing appropriate amounts of HP-beta-CD and S(-)BVC to a final 1:1 molar ratio. Differential Scanning Calorimetry (DSC) and Scanning Electron Microscopy (SEM) were used to caractereze the complex. Cytotoxic assays were performed using the tetrazolium reduction test on Balb/c fibroblasts (3T3 cells) and hemolysis was followed by the amount of hemoglobin released by human erythrocytes (0.15% hematocrit). DSC results showed that the characteristic endothermic peak of S(-)BVC (at 262.5ºC) is lost after complexation with HP-beta-CD. SEM images revealed that the characteristic crystal structures of S(-)BVC and HP-beta-CD. The hemolytic activity of S(-)BVC:HP-beta-CD was stronger than that of S(-)BVC revealing the increased interaction with membranes. S(-)BVC reduced the cell viability to 80 %, while S(-)BVC:HP-beta-CD induced effects similar to those of HP-beta-CD, without affecting cell viability up to 2 hours after treatment, as compared to S(-)BVC (p < 0.001). These results are indicative that S(-)BVC:HP-beta-CD is a less toxic formulation than S(-)BVC representing an interesting drug-delivery system. Supported by FAPESP.