Objective: To determine the macronutrient and energy content of preterm mothers’ milk in our tertiary care center for planning optimum nutrition strategies. Methodology: This prospective observational study evaluated the macronutrient and energy contents of preterm mother’s milk. The breast milk composition of 50 mothers was analyzed on 3±2, 7±2, 14±2, 21±2, and 28±2 days of birth. The carbohydrate, protein, and fat content in milk were measured by Polberger alkaline copper reduction method, Lowry method (modified by Peterson), and colorimeter method respectively, and we also studied their correlation with body mass index (BMI) of mothers. Results: Initially, median (inter-quartile range [IQR]) carbohydrate contents were 1.2 (1.0-1.6) g/dL, which increased to 2.1 (1.5-2.4) g/dL; the protein contents were 1.7 (1.2-2.2), later declined to 1.4 (1.0-2.2) g/dL; and fat contents were 0.8 (0.6-1.2), increased to 1.2 (0.7-1.5) g/dL at end of the study. The median energy contents were 23.3(20.7-29.5), increased to 28.5(25.3-33.7) kcal/dL during the study. The carbohydrate, fat, and energy contents showed a moderate correlation with the BMI of the mothers. Conclusion: Due to variable macronutrient and energy contents of the preterm mother’s milk; individualized milk fortification policies should be developed.