Fluorine‐18 (18F) is the most favorable positron emitter for radiolabeling Positron Emission Tomography (PET) probes. However, conventional 18F labeling through covalent C‐F bond formation is challenging, involving multiple steps and stringent conditions unsuitable for sensitive biomolecular probes whose integrity may be altered. Over the past decade, an elegant new approach has been developed involving the coordination of an aluminum fluoride {Al18F} species in aqueous media at a late‐stage of the synthetic process. The objective of this study was to implement this method and to optimize radiolabeling efficiency using a Design of Experiments (DoE). To assess the impact of various experimental parameters on {Al18F} incorporation, a pentadentate chelating agent NODA‐MP‐C4 was prepared as a model compound. This model carried a thiourea function present in the final conjugates resulting from the grafting of the chelating agent onto the probe. The formation of the radioactive complex Al18F‐NODA‐MP‐C4 was studied to achieve the highest radiochemical conversion. A complementary "cold" series study using the natural isotope 19F was also conducted to guide the radiochemical operating conditions. Ultimately, Al18F‐NODA‐MP‐C4 was obtained with a reproducible and satisfactory radiochemical conversion of 79 ± 3.5% (n = 5).