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In this work we introduced a new two phase freezing (TPF) method coupled with gas chromatography for the extraction, clean up and determination of methadone (MT) in human milk samples. TPF procedure was optimized for extraction of MT from immature milk sample. The extraction of MT was performed from 1.0 ml of milk that contain 0.2 ml of Briton Robinson buffer (pH= 2.5) and 0.3 ml of acetonitrile. For separation of acetonitrile from aqueous solution, the solution was placed in refrigerator at− 40 C. The MT was analyzed by gas chromatography. The results demonstrated that the amount of MT that transferred to milk is significantly different from other published reports. The immature milks of six women who were used MT (dose of 90 mg/day) in duration of 1, 2, 3, and 5 h after consumption were analyzed. Our data demonstrated that, before one hour and also 5 h after MT consumption the breastfeeding is safe …