Background Gamma‐aminobutyric acid (GABA) is an inhibitory neurotransmitter in human brains, playing a role in the pathogenesis of various psychiatric disorders. Current methods have some non‐neglectable shortcomings and noninvasive and accurate detection of GABA in human brains is long‐term challenge. Purpose To develop a pulse sequence capable of selectively detecting and quantifying the 1H signal of GABA in human brains based on optimal controlled spin singlet order. Study Type Prospective. Subjects/Phantom A phantom of GABA (pH = 7.3 ± 0.1) and 11 healthy subjects (5 females and 6 males, body mass index: 21 ± 3 kg/m2, age: 25 ± 4 years). Field Strength/Sequence 7 Tesla, 3 Tesla, GABA‐targeted magnetic resonance spectroscopy (GABA‐MRS‐7 T, GABA‐MRS‐3 T), magnetization prepared two rapid acquisition gradient echoes sequence. Assessment By using the developed pulse sequences applied on the phantom and healthy subjects, the signals of GABA were successfully selectively probed. Quantification of the signals yields the concentration of GABA in the dorsal anterior cingulate cortex (dACC) in human brains. Statistical Tests Frequency. Results The 1H signals of GABA in the phantom and in the human brains of healthy subjects were successfully detected. The concentration of GABA in the dACC of human brains was 3.3 ± 1.5 mM. Data Conclusion The developed pulse sequences can be used to selectively probe the 1H MR signals of GABA in human brains in vivo. Evidence Level 1 Technical Efficacy Stage 1