There are many instances when mothers are unable to provide their own milk, which is the case with many prematurely born infants.  Breast milk banks and facilities that process human milk provide an alternative solution to synthetic or animal derived infant formula, allowing babies to receive the benefits of human breast milk.  There are many drugs that can pass into a woman’s breast milk and cause possible harm to an infant.  It is important that donor milk be screened for drugs-of-abuse in order to prevent this from occurring.  The purpose of this study was to optimize and validate enzyme-linked immunosorbent assays (ELISA) for the detection of a seven-drug panel in human breast milk.  The following Neogen Corporation kits were utilized: Amphetamine Ultra, Benzodiazepine Group, Cocaine/Benzoylecgonine (BZE), Cotinine, Opiate Group, Oxycodone/Oxymorphone, and THC.  Sample dilutions that minimized breast milk matrix interference were determined, and cutoff levels for each assay were proposed based on the linear range of the assay.  The seven-drug panel was validated through the assessment of drift, precision, and accuracy.  The Cocaine/BZE and Opiate Group cutoffs were increased from 30 to 50 ng/mL after several false negative results were obtained during the accuracy portion of the validation.  The ELISA assays were validated at two different sites, and the robustness of the method was demonstrated.