Abstract The scope of a recently developed optical biosensor platform for application to milk products was extended in this study to the determination of folate in supplemented and non-supplemented foods. The technique utilises folate-binding protein within an automated high throughput inhibition assay format and was subjected to single-laboratory validation. Critical performance factors were evaluated, including establishing four-parameter logistic calibration parameters, confirmation of equivalent molar cross-reactivity as a function of poly-γ-glutamate chain length, demonstration of analytically insignificant non-specific binding, and verification of the absence of food-derived matrix interferences. A panel of certified reference food materials was used to estimate assay precision (HorRatr: 0.3–0.8), confirm recovery within accepted guidelines (72–112%) and establish method accuracy based on data compliance with assigned folate values. The optimised method was applied to a range of food items (milk, cereal, flour, broccoli, egg, fishmeal, liver) and the influence of assay variables including preheat and trienzyme treatments was demonstrated to be minor. The described assay is an expedient alternative to current methods used for the determination of the folate content in foods.