Analytical techniques used to quantify neurosteroids in biological samples are often compromised by non-specificity and limited dynamic range which can result in erroneous results. A relatively rapid and inexpensive gas chromatography-mass spectrometry (GC-MS) was developed to simultaneously measure nine neurosteroids, including allopregnanolone, estradiol, and progesterone, as well as 25-hydroxy-vitamin D3 in plasma samples collected from adult women subjects during and after pregnancy. Sample preparation involved solid-phase extraction and derivatization, followed by automated injection on a GC equipped with a mass selective detector (MSD) operated in single ion monitoring (SIM) mode to yield a run time of less than 11min. Method detection limits for all neurosteroids ranged from 30 to 200pg/mL (parts per trillion), with coefficients of variation that ranged from 3% to 5% based on intra-assay comparisons run in triplicate. Although concentrations of estradiol measured by chemiluminescent immunoassay (CIA) were consistent with values determined by GC-MS values, CIA yielded considerable higher values of progesterone, suggesting antibody cross reactions resulting from low specificity. Mean neurosteroid levels and representative time-course data demonstrate the ability of the method to quantify changes in multiple neurosteroids during pregnancy, including rapid declines in neurosteroid levels associated with delivery. This simplified GC-MS method holds particular promise for research and clinical laboratories that require simultaneous quantification of multiple neurosteroids, but lack the resources and expertise to support advanced liquid chromatography-tandem mass spectrometry facilities.