In the last decade, the biomedical field has benefited from the progressive development of antiviral drugs. Antiviral drugs Sofosbuvir (SOF), ledipasvir (LED) and acyclovir (ACY) are a class of medications used specifically for treating viral infections as hepatitis C (HCV) and B (HBV) viruses. Sofosbuvir (SOF) and ledipasvir (LED) in a single-tablet regimen represent a revolution in the treatment of chronic hepatitis C which inhibits viral production of HCV. The major problem with simultaneous determination of SOF, LED and ACY by electrochemical methods is the closeness of their oxidation potentials resulting in overlapped voltammetry responses. In such frame an innovative strategy is introduced, based on a novel GC/CNT/ILC/RGO/MnO2 layered electrode, obtained by modifying a glassy carbon surface with layers of multi-walled carbon nanotubes (CNT), an ionic liquid crystal (ILC), graphene (RGO) and MnO2. The layered sensor showed excellent current responses for the simultaneous electrochemical analysis of SOF, LED and ACY in human serum in the concentration ranges 0.20–150μmoldm−3, 0.0070–15μmoldm−3 and 0.010–30μmoldm−3 with low detection limit values of 9.8nmoldm−3, 0.11nmoldm−3 and 0.84nmoldm−3 respectively. Moreover, on the new layered electrode the electro-oxidation of a quinary mixture of ascorbic acid (AA), dopamine (DA), paracetamol (APAP), SOF and LED yielded five well-separated peaks. This electrochemical method is advantageous over other methods mentioned in the literature in terms of cost, time effectiveness, sensitivity, selectivity and lower detection limit. Analysis of ACY and a combination from LED and SOF in their pharmaceutical formulations were achieved with good recovery results.