Fluorescence-based molecular imaging in small animals is having a major impact on drug development and disease research and the ability to detect multiple molecular species at once is becoming increasingly important. Unlike bioluminescence, in fluorescence, ubiquitous autofluorescent signals from the skin need to be separated from that of labeled fluorophores to ensure proper quantitative data. Since its introduction in 2004, spectral imaging methods have become an important part of in vivo fluorescence imaging of small animals by enabling easy multiplexed imaging methods and through the quantitative removal of interfering skin autofluorescence signals. This article is a review of the literature on spectral imaging methods and applications in fluorescence imaging of small animals.