In vivo optical imaging can provide information at the cellular biomarker level regarding disease states and therapeutic response. Bioluminescence imaging presents few challenges with respect to imaging and data acquisition. However, fluorescence imaging requires strategies to compensate for background fluorescence. Without proper background subtraction, results may underestimate biological changes or the magnitude of therapeutic efficacy of a drug. There are several contributors to background fluorescence which can vary depending on disease model and probe used all of which need to be considered when developing a mouse model for fluorescence imaging. Learn about key considerations for defining and applying background correction to improve fluorescence quantification and interpretation using the IVIS® platform in this technical note.