Usability is considered one of the most significant aspects to contemplate during the software development process. For this reason, it is necessary to have tools that allow development teams and specialists to evaluate whether the interface design proposals are usable, understandable, and easy to use. One of the widely used instruments to determine if an interface design is usable are questionnaires. There are some proposals aimed at capturing the perception of end-users. Still, very few approaches are focused on covering all aspects of E-Commerce applications and are aimed at specialists in Human-Computer Interaction. In this study, we have used a proposal of a new questionnaire that was developed considering an in-depth analysis of the literature and the information collected from two consulting firms. This new assessment tool intended to provide specialists a way to perform more accurate usability evaluations that cover all aspects of a software in the E-Commerce domain and at the same time allows to quantify the level of user experience. The questionnaire was used to evaluate the usability of the three main E-Commerce websites in Peru. The results show that, despite being the most relevant platforms in the country, they must improve many aspects of usability. There is little concern on the part of companies in offering websites that are intuitive and that generate satisfaction to end users.