The BTI-Tn5B1–4 insect cell line, commercially available as the High Five cell line (Invitrogen), supports higher levels of recombinant protein production compared to existing insect cell lines. Proprietary serum-free media such as ExCell 405 (JRH Biosciences), Express Five (Life Technologies), IS BAC (Irvine Scientific), and CCM3 (HyClone) are available which were developed specifically for a suspension culture of High Five cells. While these media are highly optimized, a lower cost alternative is desirable for large-scale protein production which is also serum-free and supports good cell growth (>5 × 106 cells/mL) and recombinant protein production (>50 mg/L of secreted protein). The amino acid and carbohydrate metabolism of the Tn5B1–4 cells was first examined. It was found that asparagine was nearly depleted during batch growth in Ex-Cell 405, without limitations in glutamine, other amino acids, or glucose. Alanine also accumulated to about 35 mM during growth. We then extended the formulation techniques for medium development used for Spodoptera cell lines to the Tn5B1–4 cell line. A medium based on IPL-41 basal medium, Hy-Soy protein hydrolysate (Quest, International), yeastolate ultrafiltrate, a lipid−sterol emulsion, and Pluronic F-68 was developed. Dextran sulfate (100 μg/mL) was used to induce a single cell suspension culture. This medium is denoted as ISYL and performs best when supplemented with a 2.5% lipid−Pluronic F-68 mixture. Supplementation with additional aspargine in a 1.5% lipid−Pluronic F-68 mixture did not improve growth, suggesting that a lipid was growth-limiting and not an amino acid. Ex-Cell 405 and ISYL with 2.5% lipid−Pluronic F-68 supplement supported virtually identical growth rates, extent of growth (ca. 6.0 × 106 cells/mL) in an 80% oxygen atmosphere, and supported production of SEAP (secreted human alkaline phosphatase) at a volumetric level of about 65−70 mg/L. Thus, the less expensive ISYL medium can deliver acceptable performance and may be suitable for large-scale insect cell cultures.