Teacher assessment literacy plays a pivotal role in teacher education programs; however, there seems to be a lack of either assessment literacy or its implementation. Using an online assessment course, including both theoretical and practical issues, this mixed-method study examined 16 teachers’ (8 in-service and 8 pre-service) assessment literacy and the extent to which they implement this knowledge. The quantitative part explored participants’ assessment literacy, while the qualitative phase examined the validation of the quantitative results as well as the implementation of assessment literacy in the practical realm. Data were collected via valid and reliable questionnaires, one of which was adapted from Mertler (2003), and the two others were developed by the researchers, along with a practical assessment project. The results indicated that though in-service teachers were more assessment literate at the beginning due to their experience, they were at a lower degree of assessment literacy at their eventual performance in comparison with pre-service teachers. The qualitative analysis explored the lack of teachers’ preference for the use of assessment literacy in their classroom practice. The study suggests the inclusion of both theoretical and practical dimensions of assessment literacy in teacher education programs and it proposes doing an in-depth investigation into the difficulties that hinder teachers from putting their theoretical assessment knowledge into practice.