Cyclosporine is an immunosuppressant drug used in organ transplants or for the treatment of autoimmune diseases. We developed and validated a simple, sensitive, and specific method using UPLC-MS/MS to determine cyclosporine levels in human whole blood. MS/MS detection was performed in the positive electrospray ionization mode with multiple reaction monitoring. Cyclosporine was extracted from whole-blood samples using ascomycin as an internal standard. The mass transitions m/z 1203.49 → 1185.53 and m/z 814.71 → 796.67 were used to assay the analyte and IS. This method was validated with respect to linearity, specificity, accuracy, precision, recovery, and stability. The method exhibited a linear response from 10 to 1000 ng mL(-1) with correlation coefficient values >0.99. The precision and the accuracy values were within 15%, except at the lower limit of quatification (LLOQ). Cyclosporine was stable in whole blood with no evidence of degradation. This method was successfully applied to a pharmacokinetic study of cyclosporine in healthy volunteers following oral administration.