Yeastolate is an efficient serum replacement to fully support insect cell growth and baculovirus replication in the insect cell/baculovirus expression system. However, yeastolate is an undefined complex substance and therefore subject to batch-to-batch variation, which often causes inconsistent cell growth and productivity. Therefore, the present study aims to identify yeastolate components that promote insect cell growth and baculovirus production. Gel filtration chromatography was used to determine the molecular weight profiles of yeastolate and fresh/spent culture media during Sf-21 insect cell growth and infection. By comparing the differences among these molecular weight profiles, yeastolate was fractionated into five different molecular weight ranges of fractions. Yeastolate fraction with molecular weight below 1 kD was found to contain bioactive components responsible for the cell growth, but the ingredients essential for baculovirus production were localized in yeastolate fraction between 1 to 3 kD. These results indicate that nutrient utilization was significantly different during the cell growth and infection. The present study provides a high-resolution guideline for further identification of specific bioactive ingredients in yeastolate to develop fully chemically-defined serum-free media for the insect cell/baculovirus system.