A method for the simultaneous (one-step) photochemical conjugation and ⁸⁹Zr-radiolabeling of antibodies is introduced. A photoactivatable chelate based on the functionalization of desferrioxamine B with an arylazide moiety (DFO-ArN₃, [1]) was synthesized. The radiolabeled complex, ⁸⁹Zr-1⁺, was produced and characterized. Density functional theory calculations were used to investigate the mechanism of arylazide photoactivation. ⁸⁹Zr-radiolabeling experiments were also used to determine the efficiency of photochemical conjugation. A standard two-step approach gave a measured conjugation efficiency of 3.5% ± 0.4%. In contrast, the one-step process gave a higher photoradiolabeling efficiency of ∼76%. Stability measurements, cellular saturation binding assays, positron emission tomographic imaging, and biodistribution studies in mice bearing SK-OV-3 tumors confirmed the biochemical viability and tumor specificity of photoradiolabeled [⁸⁹Zr]ZrDFO-azepin-trastuzumab. Experimental data support the conclusion that the combination of photochemistry and radiochemistry is a viable strategy for producing radiolabeled proteins for imaging and therapy.