In this study, lipidomic data of pork samples with different cold chain interruption were analyzed by combining rapid evaporative ionization mass spectrometry (REIMS) with an intelligent knife (iKnife) to develop a rapid, sensitive and accurate real-time detection technique to assess pork deterioration degree during cold chain breaks. The results showed that the relative abundance of the characterized ions differed significantly across the samples (P<0.05). Temperature fluctuations during cold chain logistics of chilled meat led to lipid oxidation. Six ions were identified as potential markers, including m/z 353.270 3, m/z 507.442 59 ([FAHFA(16:1(9Z)/5-O-16:0)]-), m/z 352.286 1 ([N-palmitoyl proline]-), m/z 271.228 4 ([2-hydroxy palmitic acid]-), m/z 331.249 4 ([9,10,13-trihydroxy-octadecanoic acid]-), and m/z 309.243 8 ([9R-HODE, methyl ester]-), which could serve as markers and effective indicators for cold chain deterioration. By incorporating the typical marker m/z 353.270 3 into the REIMS scanning scope through target enhancement, the overall recognition accuracy significantly improved from 55.77% (nontargeted acquisition mode) to 97.60% (target enhancement scanning). In conclusion, the target-enhanced iKnife-REIMS method can accurately discriminate cold chain interrupted pork according to different levels of lipid deterioration, providing near real-time results, thereby serving as a new approach to analyze deteriorated pork.