Translocase I (MraY/MurX) is an essential enzyme in growth of the vast majority of bacteria that catalyzes the transformation from UDP-MurNAc-pentapeptide (Park’s nucleotide) to prenyl-MurNAc-pentapeptide (lipid I), the first membrane-anchored peptidoglycan precursor. MurX has been received considerable attentions to the development of new TB drugs due to the fact that the MurX inhibitors kill exponentially growing Mycobacterium tuberculosis (Mtb) much faster than clinically used TB drugs. Lipid I isolated from Mtb contains the C50-prenyl unit that shows very poor water-solubility, and thus, this chemical characteristic of lipid I renders MurX enzyme assays impractical for screening and lacks reproducibility of the enzyme assays. We have established a scalable chemical synthesis of Park’s nucleotide-Nε-dansylthiourea 2 that can be used as a MurX enzymatic substrate to form lipid I analogues. In our investigation of minimum structure requirement of the prenyl phosphate in the MraY/MurX-catalyzed lipid I analogue synthesis with 2, we found that neryl phosphate (C10-phosphate) can be recognized by MraY/MurX to generate the water-soluble lipid I analogue in quantitative yield under the optimized conditions. Herein, we report a rapid and robust analytical method for quantifying MraY/MurX inhibitory activity of library molecules.