A method for determination of calcium in a sample derived from a living body, by the reaction of calcium in the sample with Chlorophosphonazo-III or a compound analogous thereto in the presence of vanadate ions, followed by determination of calcium in the sample on the basis of an optical change caused by the reaction product, is excellent in various respects as follows: it is free from the problem of absorption of carbonic acid gas caused by a high pH; it is free from the problem of use of a toxic reagent containing arsenic; it makes it possible to subject a large number of samples to determination in a short time because it can be applied to an autoanalyzer; and it permits determination in a wide range because of low sample blank values.