A sensitive and reliable stripping voltammetric method was developed to determine timolol drug. This method is based on the adsorptive accumulation of the drug at a hanging mercury drop electrode (HMDE) and then a negative sweep was initiated, which yield a well defined cathodic peak at –850 mV versus (Ag/AgCl) silver reference electrode. To achieve high sensitivity, various experimental and instrumental variables were investigated such as supporting electrolyte, pH, accumulation time and potential, scan rate, frequency, pulse amplitude, convection rate and working electrode area. The monitored adsorptive current was directly proportional to the concentration of timolol and it shows a linear response in the range from 1 × 10<sup>–7</sup> to 1.5 × 10<sup>–6</sup> mol●l<sup>–1</sup> of this drug (correlation coefficient = 0.998) and the detection limit (S/N = 3) is 1.26 × 10<sup>–9</sup> mol●l<sup>–1</sup> at an accumulation time of 30 sec. The developed adsorptive stripping voltammetry (AdSV) procedure shows a good reproducibility, the relative standard deviation RSD% (n = 8) at a concentration level of 1 × 10–6 mol●l<sup>–1</sup> of timolol was 0.13%, whereas the method accuracy was indicated via the mean recovery of 110% ± 1.414%. Possible interferences by several substances usually present in the pharmaceutical formulations have been also evaluated. The applicability of this approach was illustrated by the determination of the drug in pharmaceutical preparation and biological fluids such as serum and urine