Despite the advancements in the production and accessibility of videos and animations, a gap exists between their potential for science teaching and their actual use in the classroom. The aim of this study was to develop and evaluate an approach to boost chemistry and biology teachers’ Technological Pedagogical Content Knowledge (TPACK) and their confidence regarding the use of videos and animations in class, which are required for their effective implementation. Twelve experienced high-school chemistry and biology teachers participated in a professional development workshop including biochemistry and technological–pedagogical lectures along with video-editing instruction and practice. Teachers were provided with digital videos including high-resolution scientifically based animations and were encouraged to edit them based on their pedagogical experience and the needs of their class. We investigated how the workshop affected teachers' TPACK-confidence and TPACK. TPACK-confidence was assessed by pre- and post-workshop questionnaires and open-ended feedback questionnaires. TPACK was assessed by analyses of the edited digital videos and pedagogical considerations submitted by the teachers. It was found that teachers' TPACK-confidence was significantly higher following the workshop. There was also a development in the teachers' TPACK. They were able to recommend to use digital videos in a variety of classroom situations based on the technological pedagogical knowledge (e.g., as an opening to a new topic) and their TPACK (e.g., to visualize complex biochemical processes). We also found a development in their video-editing skills and their knowledge of how to use this technology effectively in biochemistry lessons. Results indicate that training teachers in using technological tools while providing them with relevant Content Knowledge and TPACK, and relying on their pre-existing Pedagogical Content Knowledge may assist them develop their TPACK and TPACK-confidence. This may promote the effective use of videos and animations in biochemistry teaching.