Recent mechanical studies of ultrafine electrospun polymer nanofibers showed they can simultaneously possess high strength and toughness attributed to high degree of chain alignment coupled with low crystallinity. Quantitative analysis of macromolecular alignment in nanofibers is needed for better understanding of processing/structure/properties relationships and optimization. However, quantification of structural features in nanofibers with ultrafine diameters and low variable crystallinity is highly challenging. Here, we show that application of standard orientation analysis protocols developed for polarized Raman microscopy of bulk polymers and films can lead to severe errors in subwavelength diameter samples. A modified polarized Raman method is proposed and implemented for study of size-dependent orientation in individual nanofibers as small as 140 nm. Macromolecular alignment improved significantly with the reduction of nanofiber diameter, correlating with nanofiber modulus increase. Applicability of the proposed method for quantitative comparative studies of nanofiber systems fabricated from solutions with different solvents is demonstrated.