This paper reports the development of the new technique of Raman linear difference (RLD) spectroscopy and its application to small molecules: anthracene and nucleotides adenosine-5′-monophosphate, thymidine-5′- monophosphate, guanosine-5′-monophosphate, and cytidine-5′- monophosphate. In this work we also present a new alignment method for Raman spectroscopy where stretched polyethylene films are used as the matrix. Raman spectra using light polarized along the orientation direction and perpendicular to it are reported. The polyethylene (PE) film spectra are consistent with powder samples and films deposited on quartz. RLD spectra determined from the difference of the parallel and perpendicular polarized light Raman spectra are also reported. The equations describing RLD are derived, and RLD spectra of anthracene and thymidine are calculated from these equations using Density Functional Theory and assuming perfect orientation of the samples. Because of the wealth of spectroscopic information in the vibrational spectra of biomolecules together with our ability to calculate spectra as a function of orientation, we conclude that RLD has the potential to provide structural information for biological samples that currently cannot be extracted from any other method.