<p>Chemical modification of monoclonal antibodies (mAbs) and their fragments gives rise to imaging probes and targeted therapies. Depending on the isotope used, radiolabeled mAbs enable positron emission tomography (PET) and single photon emission computed tomography (SPECT) imaging and can also be applied as cytotoxic therapies. Fluorescent mAb conjugates are used for a range of preclinical applications with clinical utility for intraoperative visualization of tumors. Antibody-drug conjugates (ADCs) enhance the therapeutic efficacy of mAbs and are the topic of extensive clinical development. In all these cases, chemical modifications can significantly affect mAb tumor targeting and clearance. Whole-body imaging techniques provide crucial insights into the in vivo consequences of these changes by directly tracking antibody conjugate distribution and clearance. This review examines in vivo imaging studies that compare “parental” and “modified” mAbs imaged under identical conditions to assess the effects of the cargo itself (e.g. fluorophore, chelator, drug), as well as the chemical conjugation methods. Additionally, we also describe studies that evaluate alternative strategies, including pretargeting, Fc modifications and pre- or co-dosing strategies that seek to tune the biodistribution of a given conjugate. Overall, we highlight the critical role of imaging in characterizing the in vivo performance of mAb conjugates, underscoring how these insights can inform both therapeutic efficacy and toxicity, and enable clinical translation.</p>