There are several specific digital tools now that have transformed the way science is taught. Correspondingly, teacher education programs have changed, and now they increasingly address the development of technology-related professional content knowledge (TPACK). Owing to the use of technology in specific domains of science teaching, there is an emergent need for domain-specific TPACK questionnaires. The present study investigates the development and application of a domain-specific TPACK questionnaire for teaching human biology using digital tools in a university-based teacher education program. A quasi-experimental between-subject design was applied for 13 weeks in the four study groups (n = 155). The intervention groups worked with digital tools in human biology, while the control groups participated without specific consideration of digital tools throughout the program. We succeeded in developing a questionnaire comprising seven reliable scales. The questionnaire development procedure described herein may be applicable to other science education disciplines. By the means of linear mixed modeling, we found that all students gained professional knowledge; however, treatment × time interactions revealed that belonging to the intervention group was the main driver of TPACK progression. Comparing the treatments, we found that at post-test, the intervention group reported higher levels of TPCK, TPK, and TCK, but not CK, PK, TK, and PCK. Sequential analyses of the longitudinal data highlighted that working with digital tools on a regular basis constitutes a TPACK boost effect. This study contributes to the literature on technology integration from the perspective of biology education, where specific tools that can be assessed using a domain-specific questionnaire are used.