Purpose In recent years, there has been a significant increase in opioid-related drug use among pregnant mothers, specifically Methadone, Subutex (Buprenorphine) and Suboxone (Buprenorphine and Naloxone) resulting in increased neonatal intensive care unit (NICU) admissions for treatment of neonatal abstinence syndrome (NAS). Standard tests such as urine, meconium, and cord stat blood samples have not been shown to accurately demonstrate maternal abuse of these medications or predict the clinical course of NAS. This study aims to correlate and compare clinical symptoms of NAS with cord/ placental blood concentrations of Buprenorphine, Methadone and their metabolites. Another goal is to demonstrate the ability to correctly identify maternal abuse and concentrations of these medications. Methods The design was an observational study where cord/placental blood samples were obtained from eligible subjects. In addition to the standard cord stat test done by state, samples were analyzed using liquid chromatography mass spectrometry (LC-MS/MS) to quantify the metabolites of Buprenorphine, Norbuprenorphine and Methadone. Investigators performing the LC-MS/MS were blinded. Infants were treated on attending physician’s discretion according to clinical course. All infants were followed until discharge. Demographics and clinical course, including NICU stay, were recorded. Results A total of 19 mothers were enrolled, out of which, 15 (78.9%) mothers were on Subutex, 2 (10.5%) on Suboxone and 2 (10.5%) on Methadone. Data analysis was performed only on subjects with exposure to Subutex due to low sample size for Suboxone and Methadone subjects. Cord stat performed by the state lab was negative in 33.3% of subjects; however, 100% of the cord blood samples tested by LC-/MSMS were positive. The percentage of neonates transferred to NICU for NAS was 60% of which 67% received replacement therapy. Length of stay in NICU for treatment of NAS did not have any correlation to the concentration of the metabolites in cord blood. Pearson’s correlation coefficient (r) between duration of NICU stay and Norbuprenorhine concentration was r = -0.07 (p-value = 0.40); Buprenorphine concentration was r = -0.30 (p-value = 0.14); Norbuprenorphine-glucoronate concentration was r = -0.05 (p-value = 0.43); Buprenorphine-glucoronate concentration was r = -0.31 (p-value = 0.13). No correlation was found after adding the concentrations of all the above metabolites with NICU stay r = -0.24 (p-value = 0.19). Conclusion The cord stat result is inferior to cord/placental blood levels of drug metabolites using LC-MS/MS for diagnosing maternal substance abuse in at risk infants. No correlation was found between the concentrations of metabolites and length of stay in NICU or duration of replacement therapy. This study was limited by a small sample size.