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As the K to 12 Science program was formally implemented, interventions to enhance competence and confidence of teachers in teaching science in a spiral progression approach are main concerns. This study aims to assess the chemistry content knowledge and self-efficacy of 38 in-service teachers enrolled in a graduate program from a teacher education institution using a content knowledge test (CKT) and a self-efficacy beliefs scale (SeS) using a mixed-method approach. Quantitative findings reveal that the least mastered topics in chemistry of the teacher-respondents include solutions, chemical bonding, the mole concept, gas laws, and chemical reactions. The science teachers say they are “somewhat confident” in teaching the chemistry topics. Qualitative findings include difficulties in answering the CKT and challenges encountered in teaching chemistry using the K to 12 science curriculum. In the needs analysis, key findings in the results of focus group discussion are used to verify quantitative findings. The correlation between content knowledge and self-efficacy beliefs is r = -0.12, with findings showing a negligible to low correlation. This implies that even if teachers perceive that they are “somewhat confident” in teaching chemistry topics, such beliefs do not match their content knowledge scores. Valid findings are based on the CKT results and further suggest that the CKT (not the SeS) is a good measure in determining the content learning needs of teachers.