Background Iron deficiency anemia (IDA) is a major health concern among pregnant women, with significant implications for maternal and fetal outcomes. Iron-folate supplementation is widely recommended to address this issue, but various factors influence its effectiveness. This study aimed to evaluate the hematological and ‘biochemical responses to iron-folate supplementation in pregnant women attending antenatal’ care and identify its success determinants. Methods A prospective observational study was conducted at the Obstetrics and Gynecology Department of Hayatabad Medical Complex, Peshawar, from January 2022 to January 2023. A total of 120 pregnant women with diagnosed iron deficiency anemia were included. Participants received daily iron-folate supplementation for three months containing 60 mg of elemental iron and 400 µg of folic acid. Hematological parameters (hemoglobin, hematocrit, red blood cell indices) and biochemical markers (serum ferritin, serum iron, and transferrin saturation) were measured pre- and post-supplementation. Data were analyzed using SPSS version 26, with a p-value of <0.05 considered statistically significant. Results Keywords Hemoglobin levels increased significantly from 9.8 ± 1.2 g/dL at baseline to 11.6 ± 1.0 g/dL post-supplementation (p < 0.001). Serum ferritin levels rose from 15.3 ± 5.6 ng/mL to 42.1 ± 8.2 ng/mL (p < 0.001), indicating improved iron stores. ‘Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) also showed significant improvements’ (p = 0.03 and p = 0.02, respectively). Maternal outcomes were favorable, with a low prevalence of preterm deliveries (8%) and a mean neonatal birth weight of 2950 ± 450 g. Side effects were reported by 15% of participants, and compliance was good in 70%. Conclusion Iron-folate supplementation significantly improves hematological and biochemical parameters, reducing anemia and enhancing maternal and neonatal outcomes. Addressing adherence barriers and sociodemographic disparities is essential for optimizing the effectiveness of supplementation programs. These findings underscore the importance of strengthening antenatal care strategies to mitigate the burden of anemia in pregnant populations.