New synthetic opioids, especially fentanyl and its analogs, are causing the most recent acceleration in opioid abuse. The presence of fentanyl analogs as mixtures in illicit drugs makes it hard to estimate their potencies. This makes the detection and differentiation of fentanyl analogs critically significant. Most of the screening methods in current use have difficulty in detecting the full range of opioid analogs due to a wide variety of structural variations. However, Raman spectroscopy, specifically surface-enhanced Raman spectroscopy (SERS) is quite capable of detecting and identifying previously known and/or unknown fentanyl analogs. The SERS technique uses Raman spectroscopy combined with colloidal metal nanoparticles to yield highly sensitive SERS spectra. It can also differentiate structurally similar fentanyl analogs due to its ability to yield spectroscopic fingerprints for the detected molecules. Certain fentanyl analogs such as carfentanil, furanyl fentanyl, acetyl fentanyl, 4- fluoroisobutyryl fentanyl, and cyclopropyl fentanyl, have gained popularity and constitute 76.4 percent of the fentanyl analogs identified in drug seizures. Several of these have been already described using Raman spectroscopy. However, there are many other fentanyl analogs that are structurally similar to 4-fluoroisobutyryl fentanyl or cyclopropyl fentanyl. Thus, it is important to differentiate these analogs from similar molecules in order to track and identify trends in illicit distribution. In this presentation, we develop methods for the differentiation of structurally similar fentanyl analogs using theoretical and experimental methods. To do this, a set of fentanyl analogs were examined using Density Functional Theory (DFT) calculations. These results were then compared with Normal Raman and SERS techniques and analyzed using statistical methods. Structurally similar fentanyl analogs have been able to be differentiated from each other. The ultimate goal of the project will be to assist law enforcement in identifying and differentiating unknown fentanyl analogs individually and in drug mixtures. The experimental results obtained in this project can be readily implemented in field applications and in the smaller laboratories, where inexpensive portable Raman spectrometers are often present and are used in drug analysis.