Oxygen binding to hemoglobin in the blood and its delivery from the lung to the tissues are conceptually assessed by a new two-tier diagnostic test. This study presents the development stages and the validity evidence of the two-tier diagnostic test. The validity measure relies on seven expert opinions who are professors and actively teach- ing biochemistry courses across the United States. The remaining number of questions turns out to be 11. Besides, the analysis of the preliminary data, which is expected to be about 30 undergraduates, will provide the evidence on students’ conceptual patterns about oxygen binding to hemoglobin and its delivery to tissues. In conclusion, this study contributes to the chemistry education research by a new instrument on a difficult-to-learn biochemistry topic and its preliminary results.