Positron emission tomography (PET) using radiolabeled biomolecules is a translational molecular imaging technology that is increasingly used in support of drug development. Current methods for radiolabeling biomolecules with fluorine-18 are laborious and require multistep procedures with moderate labeling yields. The Al18F-labeling strategy involves chelation in aqueous medium of aluminum mono[18F]fluoride ((Al18F)2+) by a suitable chelator conjugated to a biomolecule. However, the need for elevated temperatures (100-120 °C) required for the chelation reaction limits its widespread use. Therefore, we designed a new restrained complexing agent (RESCA) for application of the AlF strategy at room temperature. Methods. The new chelator RESCA was conjugated to three relevant biologicals and the constructs were labeled with (Al18F)2+ to evaluate the generic applicability of the one-step Al18F-RESCA-method. Results. We successfully labeled human serum albumin with excellent radiochemical yields in less than 30 minutes and confirmed in vivo stability of the Al18F-labeled protein in rats. In addition, we efficiently labeled nanobodies targeting the Kupffer cell marker CRIg, and performed μPET studies in healthy and CRIg deficient mice to demonstrate that the proposed radiolabeling method does not affect the functional integrity of the protein. Finally, an affibody targeting HER2 (PEP04314) was labeled site-specifically, and the distribution profile of (±)-[18F]AlF(RESCA)-PEP04314 in a rhesus monkey was compared with that of [18F]AlF(NOTA)-PEP04314 using whole-body PET/CT. Conclusion. This generic radiolabeling method has the potential to be a kit-based fluorine-18 labeling strategy, and could have a large impact on PET radiochemical space, potentially enabling the development of many new fluorine-18 labeled protein-based radiotracers.