A novel method for the determination of lipid oxidation using Raman microscopy was developed. A home-made surface-enhanced Raman spectroscopy (SERS) substrate based on silver nanoparticles deposited on a glass Petri dish was used. The degradation of ground pork stored at 5 °C was monitored for 16 days. Two packages were considered: an active packaging containing an oregano extract with antioxidant properties and a conventional one consisting of a low-density polyethylene (LDPE) film. The lipid fraction of the ground pork was extracted with a mixture of diethyl ether/n-hexane (1:1). A remarkable maximum signal enhancement factor of 1.64 × 107 at 1439 cm−1 shift (and up to 8.58 × 106 at 1655 cm−1, chosen for fat oxidation assessment) was obtained with SERS compared to conventional Raman. In addition, SERS provided better discrimination among samples than the results obtained by the thiobarbituric acid reactive substances (TBARS) method. The experimental conditions for SERS were optimized and discussed.