Fat-soluble vitamins (FSVs), A, D, and E, are components of prenatal vitamin care. Previously, limited evidence existed to explain on a molecular level how maternal FSV supplementation affects the fetus during pregnancy. We developed a simplified LC-MS/MS method to simultaneously detect FSVs in maternal plasma (MP) and amniotic fluid (AF); we used this approach to investigate the correlation between FSV levels in these two matrices. With this method, we circumvented frequently used liquid-liquid extraction or solid-phase extraction methods and, instead, used simple protein precipitation with acetonitrile for sample preparation. This method displayed satisfactory linearity, intra- and inter-day imprecision, and accuracy. We validated the consistency with standard reference material 972a and 968f certification. In analysis of MP and AF samples from 50 pregnant women in the second trimester, concentrations of retinol, 25-hydroxyvitamin D3 25(OH)D3, and α-tocopherol (reflecting vitamins A, D, and E, respectively) were lower in AF than in MP. Significant positive correlations existed between MP and AF for 25(OH)D3 (r = 0.667; P < 0.001) and retinol (r = 0.393; P = 0.005), but not for α-tocopherol (r = 0.145, P > 0.05). This novel LC-MS/MS method shows prominent applicability for FSV detection and the observed correlations contribute to research on fetal development.