Background: In Indonesian the prevalence of IDA in female adolescents is high. Higher levels of hepcidin are related to non-responsiveness to oral iron supplementation. Matriptase-2 is a major enzyme which regulates hepcidin secretion. The aim of this study was to investigate whether or not female adolescents were responsive to oral iron supplementation. Methods: This was an analytical observational study with prospective cohort approach. Subjects were 173 female adolescents who studied in year 10 and 11 of secondary schools in Boyolali regency. Hemoglobin (Hb) levels were measured using the cyanmethemoglobin method and ferritin serum used ELISA. Sixty-eight IDA subjects were given ferrous-fumarate 1 tablet/day for 1 month. After treatment, Hb levels were determined using the same method and serum levels of hepcidin and matriptase-2 were detected by ELISA. All data were analyzed using independent t-test and Mann-Whitney test with 95% significance level. Results : Â Around 50% (87/173) female adolescents had IDA and 38.2% was responsiveÂ to iron supplementation (Î”Hb â‰¥ 1 g/dL) while 61.8% was non-responsive (Î”Hb < 1 g/dL). Hepcidin levels ofÂ non-responsive subjects (6.8 ng/mL) were higher than that of responsive subjects (6.4 ng/mL), but it did not reach significant difference (p=0.302). Lower levels of matriptase-2 were observed in non-responsive subjects (666.3 pg/mL), compared with responsive subjects (1133.0 pg/mL) (p=0.074). Conclusion: More than 50% of female adolescents is not responsive to oral iron supplementation with higher levels of hepcidin and lower levels of matriptase-2. Both proteins are a potential biomarker for detection of responsiveness to oral iron supplementation.