Twenty-first century skills have attracted significant attention in recent years. Students of today and the future are expected to have the skills necessary for collaborating, problem solving, creative and innovative thinking, and the ability to take advantage of information and communication technology (ICT) applications. Teachers must be familiar with various pedagogical approaches and the appropriate ways to use ICT to support the development of their students' twenty-first century skills. The technological pedagogical content knowledge (TPACK) framework provides a theoretical model for studying the ways in which teachers use ICT in education. Still, the TPACK framework faces certain difficulties, especially concerning the instruments currently used for studying TPACK. These challenges are primarily related to the psychometric properties of the instruments and areas of pedagogical knowledge. Within this paper we introduce a new TPACK questionnaire, the TPACK-21 questionnaire which is grounded on twenty-first century skills. The TPACK-21 questionnaire is validated using confirmatory factor analysis (CFA). Results provide a six factor CFA model aligning with the TPACK theoretical framework. Also, the associations among TPACK sub-constructs, and the weak and strong areas of pre-service teachers' TPACK will be discussed.