Optical spectroscopic techniques can be used to measure Ca²⁺-binding constants when the Ca²⁺-bound and free forms of the protein display a difference in, for example, the UV absorbance, CD or fluorescence spectrum, or fluorescence polarization. One may then start with the Ca²⁺-free form, titrate in Ca²⁺ stepwise, measure a spectrum or intensity at each step, and obtain the binding constants from computer fitting to the data. The best accuracy is achieved when the protein concentration is roughly the same as the dissociation constant (the inverse of the binding constant) such that there are significant populations of both bound and free forms at several titration points. This limits the useful range of such direct measurements to binding constants below 10⁶ M-1 (K_D> 1 μM), because of the practical difficulty of making buffers with less than 0.5-1 μM free Ca²⁺. For Ca²⁺-binding proteins with affinities of 10⁶ M-1 and up, one has to rely on indirect measurements. One popular such approach uses around 1 mM ethylenediaminetetracetic acid (EDTA) or ethylene glycol-bis N,N,N′,N′-tetraacetic acid (EGTA), and a much smaller amount of protein so that the free-Ca²⁺concentration is essentially controlled by the Ca²⁺-buffering capacity of EDTA or EGTA. A potential risk with such approaches is binding of EDTA or EGTA to the protein with consequences for its Ca²⁺affinity. Another type of indirect approach outlined in this chapter involves the use of a chelator whose absorbance or fluorescence is Ca²⁺dependent (1-3).