Research and development of the HOTS test instruments was carried out to measure students’ learning outcomes in the HOTS learning process on Metabolism topic at senior high school level. This study used the research and development method of the Dick & Carey systems approach model which consists of nine stages, namely: 1) assess needs to help identify learning goals; 2) conduct instructional analysis and analyze learners and contexts; 3) write performance objectives; 4) develop assessment instruments; 5) develop instructional strategies; 6) develop and select instructional materials; 7) design and conduct formative evaluation; 8) revise instruction based on the formative evaluation; and 9) design and conduct summative evaluation. The data of the students’ mastery on Metabolism topic was collected through a HOTS-based multiple-choice test related to Metabolism topic. A total of 610 research subjects were involved in this study. Content validity analysis were conducted using Aiken’s V formula and empirical validity analysis were carried out using product moment Pearson correlation. The results reveal that based on content validation, 83 out of 100 test items are valid, proven by the Aiken’s V value on the three indicators which is greater than 0.677. Based on empirical validation, in cycle I there are 60 valid test items as rcount > rtable (0.12) and sig. 2 tailed value < α 0.05. In cycle II, there are 55 valid test items as rcount > rtable (0.11) and sig. 2 tailed value < α 0.05. The 55 test items are in good quality category, thus they are ready to be implemented.