Background . In Ghana, anaemia is a severe public health problem among adolescent girls. In an attempt to deal with this phenomenon, Ghana Ministry of Health in collaboration with other development partners developed and launched weekly iron and folic acid supplementation program for adolescent girls in Ghanaian junior high schools. Therefore, the main aim of this study was to determine the level of compliance with iron and folic acid supplementation (IFAS) and its associated factors among adolescent girls in the Tamale Metropolis of Ghana. Methods . A cross-sectional study was conducted among 424 randomly sampled adolescent girls in the Tamale Metropolis of Ghana from April to July 2019 using an interviewer-administered structured questionnaire. Twenty school health coordinators were purposively selected to answer questions on the challenges they face in implementing the IFAS program at the school level. Bivariate logistic regression and multivariate logistic regression were used to determine associations and strength of associations, respectively, at a significant threshold of p 0.05 . Results . Compliance with the IFAS was low (26.2%). Adolescent girls who were aware of anaemia (AOR = 3.57 (95% CI: 1.96, 6.51) p 0.01 ), had good knowledge of anaemia (AOR = 1.82 (95% CI: 1.17, 2.81) p = 0.01 ), and had good knowledge of the IFAS program (AOR = 2.29 (95% CI: 1.47, 3.57) p 0.01 ) were significantly associated with compliance with the IFAS. The majority (60%) of the adolescent girls have ever missed taking the iron and folic acid (IFA) tablet because it was not issued to them by the teacher’s concern while about 48.3% (169) of the adolescent girls are taking the tablet because it prevents anaemia. Adolescent girls perceiving the tablet as family planning medicine (88.8%) and unavailability of water in classrooms (18.8%) were cited as the major challenges by school health coordinators. Conclusion . Compliance with the IFAS among adolescent girls was low. Level of education and occupation of mothers of adolescent girls, awareness on anaemia, and good knowledge of anaemia and of the IFAS program were significant predictors of compliance with the IFAS. Educating the adolescent girls on anaemia and benefits of the IFAS, constant supply of the IFA tablet, and engaging parents of the adolescent girls on the program will help improve the compliance level of the adolescent girls with the IFAS.