A bioorthogonal labeling approach based on the Huisgen-click reaction including the one-step radiosynthesis of two novel versatile and bifunctional labeling building blocks ([¹⁸F]AFP) [¹⁸F]12 and ([ ¹⁸F]BFP) [¹⁸F]6 with the PET radionuclide fluorine-18 is described. Optimized reaction conditions for the fully automated synthesis procedure using the TRACERlab Fx_FN module gave both piperazine derivatives [¹⁸F]6 and [¹⁸F]12 with radiochemical yields of 31 ± 9% (S.D., n = 8, d.c.) and 29 ± 5% (S.D., n = 19, d.c.), respectively, within 40 min synthesis time and high specific activities after convenient purification using silica gel cartdridges. First biological studies of both building blocks revealed a remarkable in vitro stability in rat blood as well as rat plasma over more than 60 min. Sample ligation reactions of [ ¹⁸F]6 and [¹⁸F]12 with azide and alkyne functionalized amino acid derivatives yielded the corresponding labeled triazoles in good to high RCYs. Moreover, the azide functionalized peptide 17, which is highly affine to the EphB2 receptor due to its SNEW sequence, was synthesized and successfully radiolabeled with [¹⁸F]BFP [¹⁸F]6 under relatively mild conditions yielding the corresponding triazolyl-peptide [ ¹⁸F]18.