Oxidation of low-density lipoproteins (LDL) within the sub-intimal space leads to plaque formation, the underpinning pathophysiological mechanism of atherosclerotic cardiovascular diseases. Currently available positron emission tomography (PET) tracers for atherosclerosis target inflammation and are not specific for plaque. LO1 and LO9 are laboratory-developed antibodies capable of recognising LDL. Despite their high specificity, the use of full antibodies as PET tracers is limited due to their long circulation time, which may be overcome by pretargeted labelling. This requires trans-cyclooctene (TCO) conjugation to the antibody, followed by reaction with a radiolabelled tetrazine (Tz) in vivo, which is injected after the conjugated antibody has accumulated on target. Accordingly, Tz-NODA-Al[18F]F and [18F]FpyTz, were synthesised and radiolabelled with fluorine-18 and tested against TCO. In addition, a near-infrared fluorescence (NIRF) emitting tetrazine, Tz-VT and biotinylated Tz-biotin, were synthesised to mimic the behavior of the radiolabelled analogues for in vitro and ex vivo tests. LO9 and LO1 were conjugated to TCO via lysine residues to yield LO9-TCO and LO1-TCO, respectively. Next, enzyme-linked immunosorbent assays were performed to confirm retained function. Aortic sections from high fat-fed LDL receptor deficient (Ldlr-/-) mice were coated with LO9-TCO, LO9, LO1-TCO or LO1 and then exposed to NIR-VT; demonstrating reactivity between TCO-conjugated antibody and Tz-VT and retained antibody targeting. Next, Ldlr-/- mice fed a high-fat diet for 22 weeks were injected with either TCO-conjugated antibodies followed by the radiolabelled Tz after 72 hours, or directly labelled LO1-89Zr and LO9-89Zr, and imaged with PET/CT. Ex vivo biodistribution via ɣ-counting highlighted fast clearance of the Tz. At 30 minutes post-injection, only 3% of the injected activity was circulating, whilst 20% of the directly labelled antibody remained after 72 hours. Mice injected with LO1-89Zr and LO9-89Zr or TCO-conjugated antibodies showed greater aortic uptake than controls ex vivo. Preliminary visual analysis of in vivo PET imaging of pretargeted antibodies showed co-localisation of LO9 and LO1 at bifurcations of the aorta. In this study, we have successfully developed novel constructs for the first in vivo detection of atherosclerosis with pretargeted antibodies for PET.