A novel chronoamperometric method was developed to estimate the concentration of a neutral ionophore in fixed-site, dioctyl sebacate plasticized, poly(vinyl chloride)-based, ion-selective electrode membranes. The membranes contained between 0.5 and 16 mmol/kg valinomycin. The chronoamperometric technique was used to estimate the valinomycin concentration in freshly prepared membranes and after extraction of some of the ionophore from the membranes using dioctyl sebacate. Replicate measurements indicated a relative standard deviation in the calculated valinomycin concentration of less than 10%, and these values accurately represented the true concentration of valinomycin within 10%. The method permitted an estimate of the valinomycin concentration after valinomycin was leached from a membrane. The results of preliminary experiments using heparinized dog blood suggest that blood protein adsorption does not interfere qualitatively or quantitatively with the analysis.