The use of injectable gas-filled microbubbles during ultrasound imaging is accepted as a good method to increase image contrast. Site-targeted microbubbles are expected to provide higher sensitivity and specificity than blood pool contrast agents (CAs). We have shown that covalent attachment of GRGDS peptide fragments to the surface of poly(lactic acid) CAs facilitates attachment to MDA-MB-231 human breast cancer cells in vitro. This paper examines the effect of process conditions during microbubble fabrication and ligand attachment and also changes in ligand surface density and shows that they have important effects on in vitro acoustic response and CA adhesion to breast cancer and cell lines. Use of intermittent sonication in the emulsion step, shortening of reaction times, and increase in freeze-drying times allows for a reduction of 50% in the dose of GRGDS-modified capsules (from 0.16 to 0.012 mg/mL) required to achieve a maximum enhancement of 20 dB; signal loss after 15 min insonation of GRGDS-modified capsules is reduced from a loss of 60% to a loss of 40%, and cell attachment after 10 min contact time is increased from an average of 1.4 +/- 0.86 to 1.8 +/-0.17 capsules/cell. Optimal attachment is achieved with a molar ratio of total -COOH groups to GRGDS of 1:0.5. The effect of process conditions during microcapsule fabrication, ligand attachment, and ligand surface density on in vitro acoustic response and CA adhesion to breast cancer cell lines in tissue culture are shown to be important parameters that can aid in the future design of an ultrasound CA that allows both cancer detection and treatment, potentially by targeted drug delivery.