Abstract: Rapid estimation of serum calcium levels is of pivotal importance in the diagnosis and institution of therapy for milk fever. Several laboratory methods are available for the determination of serum calcium. However, determination of calcium concentrations in serum by these methods is complicating and time consuming. The present study was conducted to develop sequestration of blood calcium by di-sodium versenate as a method for rapid estimation of serum calcium levels. The concentration of di-sodium versenate required for preventing coagulation of known amount of blood was correlated with the serum concentration of calcium using sodium chloranilate method. A simple test for the rapid estimation of serum calcium level by calcium sequestration using di-sodium versenate was developed and used in post-parturient cows for diagnosis of milk fever.