Objective: Placenta is a temporary organ that connects the developing fetus and the mother. However, it cannot protect the embryo against chromium (Cr) and nickel (Ni) exposure. Quantification of Cr and Ni in biological and ecological subjects is challenging. Thus, the first goal of this study was to provide a validated Graphite Furnace Atomic Absorption Spectrometry (GFAAS) method to determine Cr and Ni in mothernewborn specimens. The second goal was to assess the reference Ni and Cr contents in cord blood, maternal blood, and placenta samples in a population from Ankara. Material and Methods: Biological samples were collected from 100 healthy mother-newborn pairs. Metal levels were quantified by GFAAS. Method validation of this toxicological analysis was performed by the use of certified reference materials, and assessed through accuracy, precision, specificity, range, quantitation, and detection limits. Results: Mean Cr levels of maternal blood, placentas, and cord blood were 0.337±0.222 μg/L, 0.221±0.160 μg/kg, 0.121±0.096 μg/L, respectively while mean Ni concentrations were 0.128±0.093 μg/L, 0.124±0.067 μg/kg, 0.099±0.067 μg/L, respectively. The method showed linearity with excellent correlation coefficients (r2 ) for Cr (0.9994) and Ni (0.9999). Satisfactory recovery and coefficient of variation for Cr and Ni were 102.85% and 102.35%; 1.75% and 2.91%, respectively. Relative error did not exceed 3%, demonstrating the accuracy of the method. Control charts were drawn to assess inter-day stability. The predicted reference ranges for Cr and Ni concentrations in maternal blood, placenta and cord blood were: Cr 0.033-0.75 μg/L; 0.032-0.526 μg/kg; 0.031-0.309 μg/L and for Ni were 0.011-0.308 μg/L; 0.024-0.251 μg/kg; 0.066-0.209 μg/L, respectively. Conclusion: The reported reference values of biological specimens in this paper will provide complementary aid to health professionals in terms of assessment of environmental and occupational exposure. (J Turk Ger Gynecol Assoc 2021; 22: 187-95)