Abstract: Introduction: Adolescent girls are at risk of anemia. Anemia is a condition where hemoglobin levels in the blood are below normal. Based on data from the Kupang City Health Office as of March 2023, 38% (371) of 965 junior high school girls screened had anemia. One of the factors that influence the occurrence of anemia is physical activity. Aims: Analyzing the relationship between physical activity and the incidence of anemia among junior high school girls in Kupang City. Methods: The analytic observational study with cross-sectional design. The research instrument used was the IPAQ-Short Form questionnaire to measure physical activity and blood sampling using EasyTouch GcHb to measure hemoglobin levels. The sampling technique used was probability sampling technique, namely proportionate stratified random sampling and got 100 respondents. Data analysis used was univariate and bivariate analysis using the Chi-Square test. Results: Based on physical activity, out of 100 respondents, 20% were in the heavy category, 44% in the moderate category, and 36% in the light category. Based on the incidence of anemia, out of 100 respondents, 33% were anemic and 67% were not anemic. The results of bivariate analysis of the relationship between physical activity and the incidence of anemia showed a p value = 0.684 (p>0.05). Conclusion: There was no significant relationship between physical activity and the incidence of anemia among junior high school girls in Kupang City.