Transitions between protein conformations have been found to be essential to the biological function of many proteins. These conformational transitions can be observed by the effects of Forster Resonance Energy Transfer (FRET) on fluorescence emission. We present a new fluorescence lifetime analysis method called phasor trajectory analysis (PTA) which can be used to observe these conformational transitions under single molecule conditions and on the millisecond timescale. To maximize the precision obtained from the small number of photons available for this analysis, we developed a mathematical model for digital frequency domain lifetime acquisition, which was then used to derive the hardware parameters affecting precision. Using this information, we developed a new lifetime acquisition system which makes optimal use of the photons emitted by the sample, and provides two fully independent lifetime channels. This new hardware was used to observe the conformational dynamics of a calmodulin sample labeled with a FRET pair, and encapsulated within 100nm lipid vesicles. A toolbox of analysis techniques was developed for PTA, and was used to quantitatively describe the transitions and fluctuations of the conformation of calmodulin. Analysis was done in a model-free manner, and also by applying known parameters about the system to extract more specific information. Using the information obtained, a conformational model was developed to describe the dynamic behavior of calmodulin's conformation in terms of its binding with calcium. In addition, measurements conducted in the presence of a peptide derived from Ca2+/calmodulin-dependent protein kinase II were used to examine the properties of calmodulin's conformational dynamics while interacting with its binding targets.