Protein localization and dynamics both have important roles in cell signal transduction. Biochemical studies have elucidated many details about the chain of events in signal cascades, but the poor temporal resolution and absence of spatial localization in these conventional techniques make it difficult to determine the "where and when" of protein interactions. Over the past decade, imaging technologies and biological tools have developed to a point where many fundamental questions about protein activity can be addressed at the molecular level in living cells, revealing spatio-temporal information that is not provided by traditional biochemical assays. In this review, we illustrate the power of emerging fluorescence microscopy techniques to capture and quantify protein dynamics.