A sensitive and selective bioanalytical assay was developed and validated for the determination of enantiomeric molindone in human plasma using high-performance liquid chromatography–tandem mass spectrometry along with supported liquid extraction procedures. The chiral separation was evaluated and optimized on macrocyclic antibiotic type chiral stationary phases (CSPs) based on teicoplanin aglycone (Chirobiotic TAG) in polar organic, polar ionic, and reversed-phase mode chromatography, respectively. Complete baseline separation was achieved on a Chirobiotic TAG column under isocratic condition in reversed-phase chromatography. The method validation was conducted using a Chirobiotic TAG column (100 mm × 2.1 mm) over the curve range 0.100–100 ng/ml for each molindone enantiomer using 0.0500 ml of plasma sample. The flow rate was 0.8 ml/min and the total run time was 9 min. Supported liquid extraction in a 96-well plate format was used for sample preparation. Parameters including recovery, matrix effect, linearity, sensitivity, specificity, carryover, precision, accuracy, dilution integrity, and stability were evaluated. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels were RSD ≤3.4% and relative error (RE) ≤4.2% for molindone enantiomer 1, and RSD ≤2.6% and RE ≤5.0% for molindone enantiomer 2, respectively. This validated method could be used for the determination of individual molindone enantiomer in human plasma samples from clinical studies.