An alkaline hydrolysis/liquid chromatography (LC) method was developed for determination of isoflavones in ready-to-feed soy-based infant formula. The method consists of a 15 min methanol extraction, 10 min alkaline hydrolysis, HCl neutralization, gravity filtration, aqueous dilution, and 50 min LC analysis with UV detection at 262 and 250 nm to quantify 6 isoflavone analytes: daidzin, glycitin, genistin, daidzein, glycitein, and genistein. The concentration averages for 10 commercial batches (microg aglycone/g formula) were daidzein, 6.12 +/- 1.23; glycitein, 1.19 +/- 0.16; genistein, 12.8 +/- 2.35; and total, 20.1 +/- 3.61. Validation experiments demonstrated extraction completion and analyte stability to alkaline hydrolysis. Spike recoveries ranged from 97.6 to 104.1%, and a series of accuracy assessments showed that isoflavone concentration determined by the method was within 5% of the true value. The relative standard deviation values for repeatability ranged from 0.4 to 2.2% (n = 10), and from 0.3 to 2.7% (n = 4) for intermediate precision. Isoflavone peak purity was verified by comparing sample and standard peak area ratios (262/250 nm). The limits of detection and quantitation (microg/ formula) ranged from 0.02 to 0.05 and 0.08 to 0.18 microg/g, respectively. The difference between our concentrations and those reported by others in 1995-1998 is attributable to the well-established seasonal variation in soybean isoflavone levels. Although the method was applied exclusively to ready-to-feed formula in the present study, it is equally suitable for powder and concentrated liquid infant formulas.