Development of RNA interference (RNAi)-based therapeutics has been hampered by the lack of effective and efficient means of delivery. Reliable model systems for screening and optimizing delivery of RNAi-based agents in vivo are crucial for preclinical research aimed at advancing nucleic acid-based therapies. We describe here a dual fluorescent reporter xenograft melanoma model prepared by intradermal injection of human A375 melanoma cells expressing tandem tomato fluorescent protein (tdTFP) containing a small interfering RNA (siRNA) target site as well as enhanced green fluorescent protein (EGFP), which is used as a normalization control. Intratumoral injection of a siRNA specific to the incorporated siRNA target site, complexed with a cationic lipid that has been optimized for in vivo delivery, resulted in 65%±11% knockdown of tdTFP relative to EGFP quantified by in vivo imaging and 68%±10% by reverse transcription-quantitative polymerase chain reaction. No effect was observed with nonspecific control siRNA treatment. This model provides a platform on which siRNA delivery technologies can be screened and optimized in vivo.