The role of supervision in the teaching and learning process is crucial. Quality teaching occurs when a supervisor closely collaborates with students and teachers to provide mediated support for improving instructional methods in the classroom. This descriptive and correlational study attempted to determine teacher respondents' instructional supervision standards and professional knowledge and how motivation mediates the relationship between professional knowledge and instructional supervision. A researcher-made online survey questionnaire was the main tool to gather data from one hundred twenty-four (124) teacher-respondents from Ambray District in the Division of San Pablo City. Findings revealed a positive significant relationship between instructional and supervisory practices and the teachers' professional motivation and professional knowledge. The same result was obtained between the correlation of professional motivation and professional knowledge. A mediation analysis using multiple regression revealed a direct significant relationship between instructional, supervisory practices and teachers' professional knowledge. Work satisfaction and locus of control as professional motivations partially mediate these two variables. The study suggests that the school head may provide stimulation in the school setting, allowing teachers to enhance their professional competence in modern teaching-learning modalities. Finally, future researchers are encouraged to conduct studies in a local setting to show that these variables may predict the instructional and supervisory practices of school heads at the district, division, or even regional level..