Fluorescent D-amino acids (FDAAs) are D-amino acid derivatives whose side-chain terminal is covalently coupled with a fluorophore molecule. FDAAs incorporate into the bacterial peptidoglycan (PG) in live bacteria, resulting in strong peripheral and septal PG labeling without affecting cell growth. They are featured with their in-situ incorporation mechanisms which enable time-course tracking of new PG formation. To date, FDAAs have been employed for studying the cell wall synthesis in various bacterial species (both Gram-positives and Gram-negatives) through different techniques, such as microscopy, mass spectrometry, flow cytometry.