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New acyclonucleoside analogs tethered by a 1,2,4-triazole scaffold were synthesized through the condensation of 4-amino-5-(2-phenyleth-1-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2) with benzaldehyde followed by the alkylation of the resulting Schiff base (3)with 2-bromoethanol, 3-chloropropanol and/or 3-chloropropan-1,2-diol. Voltammetric studies were carried out for the analysis of 1 × 10−6 mol L−1 of the newly synthesized acyclonucleoside analogs (4–6) using square wave-adsorptive stripping voltammetry (SW-AdSV). The sharp voltammetric peak and high reduction current were recorded using a Britton–Robinson B–R pH 10 buffer at Ep = −1250 mV on the hanging mercury drop surface (HMDE) and Ag/AgCl reference electrode. Several experimental conditions were studied, such as the supporting electrolytes, the pH, and the accumulation time, as well as the potential, the scan rate, the frequency and the step potential for 4-benzylideneamino-5-(2-phenyleth-1-yl)-3-[(2,3-dihydroxyprop-1-yl)thio]-1,2,4-triazole (6). The analytical performance of the voltammetric technique was investigated through the analysis of the calibration curve, the detection limit, the recovery and the stability. The voltammetric analytical applications were evaluated by the recovery of compound (6) in the urine and plasma samples. The HPLC technique was also applied for the separation of compound (6) from interference using a C-18 (5 μm) column with UV detection at 254 nm.