Abstract Background Although numerous evidence-based and feasible interventions are available to treat pain from childhood vaccine injections, evidence indicates that children are not benefitting from this knowledge. Unrelieved vaccination pain puts children at risk for significant long-term harms including the development of needle fears and subsequent health care avoidance behaviours. Parents report that while they want to mitigate vaccination pain in their children, they lack knowledge about how to do so. An evidence-based clinical practice guideline for managing vaccination pain was recently developed in order to address this knowledge-to-care gap. Educational tools (pamphlet and video) for parents were included to facilitate knowledge transfer at the point of care. The objectives of this study were to evaluate usability and effectiveness in terms of knowledge acquisition from the pamphlet and video in parents of newly born infants. Methods Mixed methods design. Following heuristic usability evaluation of the pamphlet and video, parents of newborn infants reviewed revised versions of both tools and participated in individual and group interviews and individual knowledge testing. The knowledge test comprised of 10 true/false questions about the effectiveness of various pain management interventions, and was administered at three time points: at baseline, after review of the pamphlet, and after review of the video. Results Three overarching themes were identified from the interviews regarding usability of these educational tools: receptivity to learning, accessibility to information, and validity of information. Parents’ performance on the knowledge test improved (p≤0.001) from the baseline phase to after review of the pamphlet, and again from the pamphlet review phase to after review of the video. Conclusions Using a robust testing process, we demonstrated usability and conceptual knowledge acquisition from a parent-directed educational pamphlet and video about management of vaccination pain. Future studies are planned to determine the impact of these educational tools when introduced in clinical settings on parent behaviors during infant vaccinations.