Optical coherence tomography (OCT) is a three-dimensional optical imaging technique that has been successfully implemented in ophthalmology for imaging the human retina, and in studying animal models of disease. OCT can nondestructively visualize structural features in tissue at cellular-level resolution, and can exploit contrast agents to achieve molecular contrast. Photothermal OCT relies on the heat-producing capabilities of antibody-conjugated gold nanoparticles to achieve molecular contrast. A pump laser at the nanoparticle resonance wavelength is used to heat the nanoparticles in the sample, and the resulting changes in the index of refraction around the nanoparticles are detected by phase-sensitive OCT. Lock-in detection of the pump beam amplitude-modulated frequency and the detector frequency allow for high-sensitivity images of molecular targets. This approach is attractive for nondestructive three-dimensional molecular imaging deep (approximately 2 mm) within biological samples. The protocols described here achieve a sensitivity of 14 parts per million (weight/weight) nanoparticles in the sample, which is sufficient to differentiate EGFR (epidermal growth factor receptor)-overexpressing cells from minimally expressing cells in three-dimensional cell constructs.