Designing stable hetero-oligomeric protein complexes with defined intersubunit stoichiometry remains a significant challenge. In this study, we report the design of a highly selective heterotrimeric assembly derived from the well-known foldon homotrimer. We generated an aab heterotrimer by replacing glutamine 11 with glutamic acid, to destabilize the homotrimer, and replacing valine 14 with either alanine or leucine, to stabilize the hydrophobic core of the heterotrimer. Native mass spectrometry (MS) was essential for guiding the design process, enabling precise characterization of oligomeric states and their equilibrium distributions. The heterotrimer structure was validated by X-ray crystallography. Our findings highlight the effectiveness of combining rational design with native MS to develop specific hetero-oligomeric assemblies.