Analytical methodologies were developed based on the near infrared spectroscopy (NIR) associated with chemometric tools to predict the content of macronutrients (total carbohydrate, total protein, and total lipids) in medical food. We prepared the samples employed in this study according to a mixture design with percentual variations from 25% to +25% of macronutrients. Partial least squares (PLS) regression models were built with 70 and 30 samples as calibration and validation sets, respectively. We chose the best processing techniques and results based on the parameters of the ASTM 1655 standard. The models were used to determine these macronutrients in marketed enteral nutrition products, of three different batches, that have quality certification. The average errors in the determination of total carbohydrate, total protein, and total lipids were 1.8, 4.3, and 2.3 %, respectively. The percentages of errors found in this study suggest that the developed model may be useful for the quality control laboratory routine.