A main challenge in chemotherapy is to determine the in cellulo parameters modulating the drug concentration required for therapeutic action. It is absolutely urgent to understand membrane permeation and intracellular concentration of antibiotics in clinical isolates: passing the membrane barrier to reach the threshold concentration inside the bacterial periplasm or cytoplasm is the pivotal step of antibacterial activity. Ceftazidime (CAZ) is a key molecule of the combination therapy for treating resistant bacteria. We designed and synthesized different fluorescent CAZ derivatives (CAZ*, CAZ**) to dissect the early step of translocation-accumulation across bacterial membrane. Their activities were determined on E. coli strains and on selected clinical isolates overexpressing ß-lactamases. The accumulation of CAZ* and CAZ** were determined by microspectrofluorimetry and epifluorimetry. The derivatives were properly translocated to the periplasmic space when we permeabilize the outer membrane barrier. The periplasmic location of CAZ** was related to a significant antibacterial activity and with the outer membrane permeability. This study demonstrated the correlation between periplasmic accumulation and antibiotic activity. We also validated the method for approaching ß-lactam permeation relative to membrane permeability and paved the way for an original matrix for determining " Structure Intracellular Accumulation Activity Relationship " for the development of new therapeutic candidates. With the misuse and overuse of the various antibiotic families and the scarcity of new drugs available, multid-rug resistance is now a major bacterial threat to healthcare worldwide 1–4. A major concern discussed in various studies is the intracellular concentration of the antibiotic molecule 5–7. In Gram-negative bacteria, the challenge for a drug is to permeate the outer membrane (OM) that protects the cell against external attacks 8, 9. This membrane plays a key role in controlling the diffusion of external toxic molecules and the antibiotics must translocate through the outer membrane to reach the periplasmic target or additionally pass the inner membrane to reach the cytoplasmic target 10, 11. During their translocation, the molecules can use various hydrophilic channels such as porins that form trimeric hydrophilic pores present in the OM 7, 8. Today a key challenge for hospital microbiologists is to bypass the resistance in a Gram-negative strain that can produce carbapenemase and/or ß-lactamase 12, 13. Recent studies proposed an alternative way by using a combination of ceftazidime (CAZ) + ß-lactamase inhibitor 14–16. Regarding this strategy, it is urgently required to dissect the relationships between the role of membrane and enzymatic barrier and the antibacterial activity of CAZ as recently reported 17. Presently, various groups have studied the binding of ß-lactams to penicillin binding proteins (PBP) using fluorophore-conjugated-penicillin, termed Bocillin 18, 19 , fluorescent cephalosporin C 20 or fluorescent meropenem 21. Despite these various efforts to develop labeled antibiotic probes, no key information has been obtained regarding the role of porins, penetration rate, and the effect of ß-lactamase on periplasmic concentrations when addressing the choice of combination of clinically-used cephalosporin and ß-lactamase