OBJECTIVE: To conduct a cost-benefit analysis of Early Start, an integrated prenatal intervention program for stopping substance use in pregnancy. METHODS: A retrospective cohort study was conducted of 49,261 women who had completed prenatal substance abuse screening questionnaires at obstetric clinics and who had undergone urine toxicology screening tests. Four study groups were compared: women screened and assessed positive and followed by Early Start (screened-assessed-followed, n=2,032), women screened and assessed positive without follow-up (screened-assessed, n=1,181), women screened positive only (screened-positive-only, n=149), women in the control group who screened negative (control, n=45,899). Costs associated with maternal health care (prenatal through 1 year postpartum), neonatal birth hospitalization care, and pediatric health care (through 1 year) were adjusted to 2009 dollars. Mean costs were calculated and adjusted for age, race, education, income, marital status, and amount of prenatal care. RESULTS: Screened-positive-only group adjusted mean maternal total costs ($10,869) were significantly higher than screened-assessed-followed, screened-assessed, and control groups ($9,430; $9,230; $8,282; all P<.001). Screened-positive-only group adjusted mean infant total costs ($16,943) were significantly higher than screened-assessed-followed, screened-assessed, and control groups ($11,214; $11,304; $10,416; all P<.001). Screened-positive-only group adjusted mean overall total costs ($27,812) were significantly higher than screened-assessed-followed, screened-assessed, and control groups ($20,644; $20,534; $18,698; all P<.001). Early Start implementation costs were $670,600 annually. Cost-benefit analysis showed that the net cost benefit averaged $5,946,741 per year. CONCLUSION: Early Start is a cost-beneficial intervention for substance use in pregnancy that improves maternal-infant outcomes and leads to lower overall costs by an amount significantly greater than the costs of the program.