A recurring theme throughout this volume is how to accurately extract quantitative information from microscope images, particularly from fluorescence images. The reason is that, in an age of dramatic pseudocolor images, it is easy to mislead first oneself, and then, in turn, others. The accurate application of correct quantitative technique in fluorescence microscopy requires careful attention to detail. Our goals in this chapter are fourfold. First, we will consider the fundamental physics of fluorescence. Second, we will begin to look at some of the issues important to quantitative measurement of fluorescence. Although quantitative measurement presents many pitfalls to the beginner, it also presents significant opportunities to one skilled in the art. Third, we will examine how fluorescence is measured in the steady state and time-domain. Finally, we will examine how fluorescence is applied in the modern epifluorescence microscope.