The pH inside drug delivery systems influences directly the physical and chemical behavior of its ingredients specifically their solubility and stability. These properties significantly affect the release performance of the formulations as well as the pharmacological effect. Therefore, the determination of the microclimate pH (μpH) inside the drug delivery systems is of great importance and interest.Implants are considered to be attractive parenteral drug delivery systems used for the long-term application of drugs and of peptides. Poly(lactide-co-glycolide) (PLGA) is the most frequently used and extensively researched polymer for implant preparation. However it is known that the microclimate pH (μpH) within the PLGA implants can also drop dramatically. This pH drop can cause peptide or protein instabilities as well as drug insolubilities and further decomposition. Although the internal pH behavior of PLGA implants and microparticles has been studied in vitro, no data about the μpH behavior in in situ forming implants has yet been described. This is due to the fact, that there is no reliable non-invasive method available to measure directly and continuously the pH in vivo. Therefore, the question if in vitro measurement results are potentially assignable remains unclear.In this study, the μpH of in situ forming PLGA implants were mapped in vitro, in vivo, and ex vivo. A non-invasive in vivo pH measurement method using the multispectral Maestro fluorescence imaging system was developed. The in vivo experiments performed, not only enabled the authors of this article to make certain assumptions about μpH behavior but also emphasized certain expectations regarding the solvent replacement in the core area of the implant as well as the release profile of hydrophilic substances.The experiments emphasized the broad application range of the fluorescence imaging technique for non-invasive monitoring of μpH values in drug delivery systems in vivo.