Introduction

. Herpes Simplex Virus type I (HSV-1) has a high prevalence, incidence, and morbidity worldwide. The usual treatment employs nucleoside analogues; however, its prolonged use has led to viral resistance, prompting the search for new therapeutic alternatives. In this context, in vitro preclinical assays in the formulation processes offer advantages such as variable control, standardization, and simplicity compared to in vivo studies, although there is limited evidence regarding their use in the evaluation of topical antiviral formulations. Objective. To evaluate a method for determining the in vitro safety and efficacy of formulations against HSV-1. Materials and Methods. Creams, gels, and ointments were formulated with 5% acyclovir as the reference active ingredient. Cytotoxicity was assessed using Resazurin, and antiviral activity was evaluated by quantifying the viral genome through qPCR. Results. The ointment showed a CC50 of 495 µg/mL, IC50 of 3.6 µg/mL, and an SI of 137.5. The gel showed a CC50 of 582.4 µg/mL, an inhibitory concentration 50 ˂1.2 µg/mL, and a selectivity index SI ˃465. Conclusion. The in vitro system based on cytotoxicity and viral quantification by qPCR was effective for evaluating topical formulations. The values obtained were within expected ranges, highlighting the method’s potential as a preliminary tool to optimize and simplify the design of subsequent in vivo studies.