In this paper we review recent applications of high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), ultra high performance liquid chromatography coupled with high-resolution time-of-flight mass spectrometry (HRTOF-MS), and matrix assisted laser desorption ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTMS) to determine penicillin residues in milk and dairy products and their major enzymatic metabolites in vivo. This review describes recent progress in the development of sample extraction, purification, chromatographic separation and mass spectrometry detection methods for the determination of penicillin residues in dairy products.