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The freshness changes in poultry fillets during storage were studied using a portable fiber-optic Raman spectrometer. Poultry fillets with the same storage life (9 days) and expiry date were purchased from a local store and stored at 4 °C. Their Raman spectra were measured on a daily basis up to day 21 using a QE Pro-Raman spectrometer with a laser excitation wavelength of 785 nm. The complex spectra were analyzed using Principal Components Analysis (PCA), which resulted in a separation of the samples into three quality classes according to their freshness: fresh, semi-fresh, and spoiled. These classes were based on and similar to the information inferred from the product label on the packages of poultry fillets. The PCA loadings revealed a decrease in the protein content of the poultry meat during spoilage, an increase in the formation of free amino acids, an increase in oxidation of amino acid residues, and an increase in microbial growth on the surface of the poultry fillets, as well as revealing information about hydrophobic interaction around the aliphatic residues. Similar groupings (fresh, semi-fresh, and spoiled) were also obtained from the results of an Agglomerative Hierarchical Cluster Analysis (AHCA) of the first five principal components. The results allow the conclusion that the portable fiber-optic Raman spectrometer can be used as a reliable and fast method for real-time freshness evaluation of poultry during storage.