In this paper a new electrochemical method for determination of the β-carboline alkaloids, harmalol and harmine, using differential pulse voltammetry is presented. Experimental parameters such as pH, scan rate, pulse amplitude and pulse time were optimized to characterize their electrochemical behavior and to find best analytical conditions. Thus, in the presence of ascorbic acid, uric acid, dopamine, the developed method exhibits excellent performance for the determination of harmalol and harmine for a linear concentration range between 1–50 μM and 1–75 μM with a detection limit of 0.6 μM and 0.2 μM, respectively. The proposed method was suc- cessfully applied for the determination of these alkaloids in a sample of an Ayahuasca liana (Banisteriopsis caapi) and in model human urine samples with good recoveries.