A growing body of mainly cross-sectional evidence suggests an association between mycotoxins, particularly aflatoxin exposure, and poor linear growth in children. We describe the design and methods of a rigorous longitudinal birth cohort study aimed to deepen our understanding of this hypothesized relationship and to validate dried blood spots as a less invasive, low-cost collection method for venous blood samples. The AflaCohort study was conducted in Banke district of Nepal from 2015 to 2019. A total of 1,675 pregnant women ages 16-49 were recruited from 17 Village Development Committees of the district. The research team collected maternal and child anthropometry data at birth and every 3 months from birth through the first year of life. Children were revisited at 18-22 months and 24-26 months of age. Questionnaires administered at the household level assessed risk factors for aflatoxin exposure and poor linear growth. One maternal venous blood sample was collected during gestation and child blood samples were collected at 3, 6, 12 and 18-22 months of age to assess concentrations of aflatoxin B1 (AFB1)-lysine adduct. One breast milk sample was collected from mothers when the child was 3 months of age to assess levels of aflatoxin M1. Serum ochratoxin A and urinary levels of fumonisin B1 and deoxynivalenol were measured at 18-22 months of age. Environmental enteric dysfunction was assessed using a lactulose:mannitol (L:M) test at 18-22 months of age. The study collected dried blood spots from a subset (n≈1200) of mothers and children to compare AFB1 concentrations with those found in matching venous blood samples. Biomarker assessments were conducted using a high-performance liquid chromatograph method. Findings from the study will help identify certain factors that warrant interventions to reduce aflatoxin-related stunting in Nepal. This study was registered at ClinicalTrials.gov as NCT03312049.