During preparative reversed-phase chromatography of recombinant human insulin-like growth factor-I (IGF), the separation of IGF from IGF aggregates cannot be determined using UV absorbance. An on-line reversed-phase chromatographic assay was developed that provides a quantitative measurement of IGF and IGF aggregates every 4 min, allowing real-time control of purified IGF collection. Process control using the on-line assay is a reliable and accurate method to collect purified IGF.