Chemotherapy is commonly used in the treatment of cancers. However, the mechanism of action of many of these agents is not well understood. We present the synthesis of a two-photon fluorophore (C625) and its biological application when chemically linked to a chemotherapeutic agent (AN-152). By using two-photon laser-scanning microscopy, the drug:fluorophore conjugate can be observed directly as it interacts with receptor-positive cell lines. The results of this project visually show the receptor-mediated entry of AN-152 into the cell cytoplasm and subsequently into the nucleus. These observations will allow for better understanding of the drug’s therapeutic mechanism, which is a subject of ongoing research aimed at improving present methods for cancer therapy.