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Sickle cell disease (SCD) results from a sequence defect in the β‐globin chain of adult hemoglobin (HbA) leading to expression of sickle hemoglobin (HbS). It is traditionally diagnosed by cellulose‐acetate hemoglobin electrophoresis or high‐performance liquid chromatography. While clinically useful, these methods have both sensitivity and specificity limitations. We developed a novel mass spectrometry (MS) method for the rapid, sensitive and highly quantitative detection of endogenous human β‐globin and sickle hβ‐globin, as well as lentiviral‐encoded therapeutic hβAS3‐globin in cultured cells and small quantities of mouse peripheral blood. The MS methods were used to phenotype homozygous HbA (AA), heterozygous HbA–HbS (AS) and homozygous HbS (SS) Townes SCD mice and detect lentiviral vector‐encoded hβAS3‐globin in transduced mouse erythroid cell cultures and transduced human CD34+ cells after erythroid differentiation. hβAS3‐globin was also detected in peripheral blood 6 weeks post‐transplant of transduced Townes SS bone marrow cells into syngeneic Townes SS mice and persisted for over 20 weeks post‐transplant. As several genome‐editing and gene therapy approaches for severe hemoglobin disorders are currently in clinical trials, this MS method will be useful for patient assessment before treatment and during follow‐up. [ABSTRACT FROM AUTHOR]