Background: When children are considered for research participation, international guidelines suggest that they should provide assent in addition to the consent of their parents or legally authorised representatives. Guidelines indicate that the assent-capable age of children, as a reference, is above seven years. However, previous studies that have clarified the appropriate age for assent are unavailable in Japan. If it can be shown that children under the age of seven have capacity for assent, it will clarify that assent is possible above the age of seven years. Therefore, we aimed to develop an instrument to assess children’s capacity for assent among four- to six-year-olds and research their capacity for studies with no invasion or intervention. Methods: To develop the instrument, we referenced a semi-structured interview instrument for assessing decision-making capacity to consent to participation in human subject research. Our instrument is composed of three sections based on 10 question items. Each answer is scored 0–2, and the total score is in the range of 0–20. Assent information was modified to be understood by four-year-olds in Japanese. To examine an instrument and to assess children’s capacity for assent, we conducted a study with 68 participants, aged to four to six years. The scores were analysed using a factor analysis, reliability analysis, one-way analysis of variance, t-test, and χ2 test.Results: The average age of participants was 5.4 years, the average interview time was 9.6 min, and the average scale score (range 0–20) was 14.4±3.8 points in total. Results showed a significant difference between four-year-olds and five-year-olds in the scale scores of assent capacity, largely attributable to differences in understanding and appreciation. Conclusions: The reliability and validity of this scale was indicated as sufficient. For studies with no invasion or intervention, five-year-olds may have the capacity for assent if provided adequate information about the research, tailored to their level of maturity with a child-friendly assent process. However, it cannot be stated that five to six-year-olds fully understand the significance of participating in a study that includes potential benefits for someone other than themselves. Future research may consider comparing six and seven-year-olds or older children.