The present study compares two methods for the determination of fetal lung maturity: the novel intrinsic fluorescence polarization ratio (IFPR) and the commercial TDx-FLMII. Amniotic fluid (AF) samples were collected from 69 women during the second and third trimesters of singleton pregnancies. Thirty-three samples were tested for IFPR only after centrifugation, and the rest were examined both before and after centrifugation. Of the latter 33 samples, 29 were assessed for lung maturity with the TDx-FLMII method as well. The results showed that IFPR values decreased with the advance in gestational age (r = 0.77, p < 0.05, n = 69). A significant correlation was found between IFPR of centrifuged and noncentrifuged samples (r = 0.94, p < 0.05, n = 36). A significant correlation was demonstrated between IFPR and TDx-FLMII values of centrifuged (r = 0.75, p < 0.05, n = 29) and noncentrifuged (r = 0.63, p < 0.05, n = 29) samples and moreover, samples considered mature by TDx-FLMII had low values of IFPR (n = 10). It can be concluded that the IFPR method can utilize noncentrifuged AF, thus suggested as a potential noninvasive method.