Simple, rapid and sensitive spectroscopic methods were developed and validated for the determination of antiviral agents, namely, penciclovir (PCV) and entecavir (ETV). The first method is based on measuring the native fluorescence of each of the cited drugs at its optimum excitation and emission wavelengths. The fluorescence intensity was measured for PCV & ETV at 363 nm and 370 nm upon excitation at 260 nm and 254 nm, respectively. The calibration curves were linear over the concentration range 0.1-0.8, 0.025-0.4 lg ml À1 for PCV and ETV, respectively. The second method is based on measuring the amplitude of ETV in the fourth derivative with Dk = 8 and scaling factor = 100 at 256.4 nm at which its acidic degradation showed zero reading over the concentration range 5-60 lg ml À1 . The proposed methods were applied for the determination of the cited drugs in bulk and pharmaceutical preparations. ICH guidelines were used for method validation. Ó 2016 Production and hosting by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).