This study aimed to develop a Biology student worksheet based on Project-Based Learning (PjBL) integrated with Science, Technology, Engineering, and Mathematics (STEM) on the human respiratory system and to determine its validity, practicality, and effectiveness in improving students’ critical thinking skills. The study employed a research and development approach using the 4D model consisting of Define, Design, Develop, and Disseminate stages; development was conducted up to the Develop stage. The needs analysis involved a Biology teacher and Grade XI students, followed by expert validation, practicality testing, and effectiveness testing. The effectiveness test involved 25 Grade XI students, while 10 students participated as representatives in the student practicality response. Data were collected using content, media, and instrument validation sheets, teacher and student response questionnaires, and pretest-posttest questions aligned with critical thinking indicators. Content validation reached 95.83%, media validation 89.58%, and instrument validation 95.00%. Practicality testing showed a teacher response of 91.66% and a student response of 96.63%. The mean pretest score of 50.20 increased to 91.56 on the posttest, with an N-gain score of 0.83 (82.65%), categorized as high. These findings indicate that the PjBL-STEM Biology student worksheet is valid, practical, and effective in supporting students’ critical thinking skills on the human respiratory system. The product has practical implications for Biology teachers seeking a structured resource for contextual, collaborative, and project-oriented learning, while further studies are needed to test its implementation in broader settings.