Targeted ultrasound contrast agents (UCA) were conjugated with biotinylated ligands, which allow them to adhere to specific diseased sites of interest. The overall adhesion of contrast agents is influenced by both the flow mediated gene expression and the local hydrodynamic forces acting on the contrast agents’ trajectories. The ability to bind under pulsatile flow conditions and scatter sound at high frequencies is important for potential applications involving intravascular ultrasound. Human aortic endothelial cells (HAECs) in a flow chamber were exposed to a steady shear pretreatment over 12 h using a peristaltic pump system and subsequent pulsatile waveforms. To stimulate an inflammatory reaction in the HAECs, cells were subsequently exposed to rhTNF945; (Roche Pharmaceuticals). Different flow rates were applied, and the binding efficacy of targeted UCA was evaluated. Real‐time transendothelial electrical impedance measurements and simultaneous acoustic measurements were performed on the same specimen with a scanning acoustic microscope. Applications related to targeting plaque in blood vessels are discussed. [This work was supported by the National Institutes of Health Grants NIH 2 P20 RR016453‐05A1 and NIH 2 G12 RR0030161‐21.]