The surface antigens CSPG4 and CD271 have been identified as possible markers for a population of therapy-resistant and tumor-maintaining cancer stem cells within triple-negative breast cancer (TNBC) and malignant melanoma, respectively. Photochemical internalization (PCI) is a novel drug delivery technology developed for high temporal-spatial controlled delivery of drugs that are normally unable to reach their target within cells. Five breast cancer cell lines, in which three were TNBC/CSPG4-expressing, as well as two CSPG4-expressing malignant melanoma cell lines were treated with PCI of the CSPG4-directed immunotoxin 225.28-saporin. In addition, two malignant melanoma cell lines, one CD271-expressing, were treated with PCI of the CD271-directed immunotoxin ME20.4-saporin. PCI enhanced the cytotoxic effect of the immunotoxins in a synergistic manner and was substantially more cytotoxic compared to PCI of streptavidin-saporin in the antigen-positive cell lines. The antigen-negative cell lines had no advantage of the immunotoxins, and in one cell line, the cytotoxic effect by PCI of 225.28-saporin could be blocked with the addition of a 20-fold excess of 225.28-mAbs. This study provides proof-of-principle that TNBC and malignant melanoma can be efficiently and selectively targeted in vitro using PCI of an immunotoxin specific for CSPG4, and is the first of its kind to demonstrate targeting of CD271 by PCI-induced delivery of ME20.4-saporin. The present work provides an important foundation for future PCI-based therapy targeting CSPG4 and CD271.