In recent years, Huanglongbing (HLB), or citrus greening disease, has devastated citrus crops throughout the world. Penicillin G has been used to treat HLB infected trees with promising results. However, the metabolites produced from the degradation of penicillin G are known to cause potentially life-threatening allergic reactions; therefore, the concentration and presence of the metabolites must be carefully monitored. We have built and revised an analytical method based on Ultra High Performance Liquid Chromatography in combination with Tandem Mass Spectrometry (UHPLC-MS/MS) in order to identify and quantitate penicillin G and its major metabolites, penillic acid and penilloic acid, in citrus fruit and juice. Here, we discuss the chromatographic conditions and revisions that improved the precision and accuracy of our measurements.