A significant challenge for solid tumor treatment is ensuring that a sufficient concentration of therapeutic agent is delivered to the tumor site at doses that can be tolerated by the patient. Biomolecular targeting can bias accumulation in tumors by taking advantage of specific interactions with receptors overexpressed on cancerous cells. However, while antibody-based immunoconjugates show high binding to specific cells, their low dissociation constants ( K) and large Stokes radii hinder their ability to penetrate deep into tumor tissue, leading to incomplete cell killing and tumor recurrence. To address this, we demonstrate the design and production of a photo-cross-linkable affibody that can form a covalent bond to epidermal growth factor receptor (EGFR) under near UV irradiation. Twelve cysteine mutations were created of an EGFR affibody and conjugated with maleimide-benzophenone. Of these only one exhibited photoconjugation to EGFR, as demonstrated by SDS-PAGE and Western blot. Next this modified affibody was shown to not only bind EGFR expressing cells but also show enhanced retention in a 3D tumor spheroid model, with minimal loss up to 24 h as compared to either unmodified EGFR-binding affibodies or nonbinding, photo-cross-linkable affibodies. Finally, in order to show utility of photo-cross-linking at clinically relevant wavelengths, upconverting nanoparticles (UCNPs) were synthesized that could convert 980 nm light to UV and blue light. In the presence of UCNPs, both direct photoconjugation to EGFR and enhanced retention in tumor spheroids could be obtained using near-infrared illumination. Thus, the photoactive affibodies developed here may be utilized as a platform technology for engineering new therapy conjugates that can penetrate deep into tumor tissue and be retained long enough for effective tumor therapy.