<p>Ultrasound is the second most common clinical imaging modality. Ultrasound image quality often suffers from poor contrast resolution and thus can greatly benefit from appropriate contrast agents. While micron-sized gas core particles (microbubbles) are clinically utilized in this space for applications such as echocardiography, a smaller agent could be more broadly applicable to enhance the sensitivity and specificity of disease detection. To this end, lipid-shelled nanobubbles have recently emerged as robust ultrasound contrast agents for both diagnostic and therapeutic purposes. They have been used in preclinical applications ranging from oncology to endocrinology and are notably relevant in diseases that involve pathological vasculature. Owing to their shell composition, nanobubbles are versatile and can be functionalized with fluorophores, targeting agents and therapeutic moieties. Here we provide the steps needed to formulate plain, fluorophore-conjugated, ligand-conjugated, hydrophilic dye-integrated and drug-loaded nanobubbles. The typical formulation for lipid-shelled nanobubbles takes place via self-assembly driven by mechanical agitation, followed by differential centrifugation. The process is deceptively simple, yet there are many nuances in the formulation process that must be followed to produce consistently successful nanobubble batches without contamination from microbubbles. Even minor deviation from the standard protocol can substantially affect nanobubble yield, stability, acoustic performance and batch-to-batch reproducibility. This procedure also details common pitfalls and their potential consequences for bubble quality and performance. The procedure requires 3 h to complete the formulation and activation of the plain nanobubbles by users with basic laboratory expertise.</p>