Gas filled microbubbles (MBs) stabilized by a shell (e.g. lipids) are commonly used as ultrasound (US) contrast agents. Attaching biomolecules to the surface of MBs allows for molecular US imaging of various diseases. With the increased interest in targeted US imaging, new platforms to prepare disease-targeted MBs are necessary. Furthermore, attaching signaling agents to MBs creates multi-modal imaging opportunities, enhancing visualization and quantification of disease biomarkers. In this thesis, MBs labeled with 99mTc and/or rhodamine dye by taking advantage of the strong interaction between biotin and streptavidin are reported. Radiolabeling of MBs was achieved in good radiochemical yield (~ 30%). 99mTc-labeled MBs were targeted to vascular endothelial growth factor receptor 2 (VEGFR2) using an anti-VEGFR2 antibody and to prostate specific membrane antigen (PSMA) using small-molecule based PSMA inhibitors. In vitro evaluations showed successful binding of MBs to the target while in vivo targeting assessments were unsuccessful. New strategies to target MBs to the site of interest were then developed through the use of the bioorthogonal reaction between tetrazine (Tz) and trans-cyclooctene (TCO). A biotinylated derivative of Tz was loaded on streptavidin coated MBs to create a Tz-derivatized MB (MBTz). Targeting MBTz to extracellular markers of cancer such as VEGFR2, PSMA and urokinase plasminogen activator receptor (uPAR) in vitro was achieved using TCO-conjugated antibodies. In vivo targeting was successful for VEGFR2 and PSMA, but not uPAR. Translating the new strategy to other US contrast agents was then investigated. Gas vesicles (GVs) produced in halobacteria were conjugated with TCO using amide-coupling chemistry. A 99mTc-labeled derivative of Tz was loaded on TCO-GVs (RCY= 59%) and their distribution assessed by SPECT/CT imaging and ex vivo tissue counting. Having established a convenient platform to conjugate molecules to GVs and MBs, future work focuses on developing a new generation of human compatible molecular US imaging probes. / Dissertation / Doctor of Philosophy (PhD)