In the present study, we introduced a novel, reliable, sensitive and highly precise capillary zone electrophoresis (CZE) method for simultaneous separation and determination of nalbuphine hydrochloride (NLB) and its related antagonist compounds naloxone hydrochloride (NLX) and naltrexone hydrochloride (NLT) in bulk drug, pharmaceuticals and human biological fluids. The separation conditions using CZE were optimized and the separation process was carried out using fused silica capillary of total and effective lengths 57 and 50 cm, respectively. The applied running buffer was acetate buffer 20 mmol L-1 of pH=3.8 at a potential 15 kV. The detection of samples was carried out using Diode array (DAD) at 230 nm with hydrodynamic injection 6 s, under 70 mbar and capillary cartridge 25°C. The applied internal solution (IS) was phenylethylamine (PEA). The developed method displayed an excellent separation of the investigated drugs with linear concentration ranges of 5-200, 20-240 and 10-280 μg mL-1 for NLB, NLX and NLT, respectively. Low detection limits were recorded as 0.3, 5.0 and 6.5 μg mL-1 while, the quantification limits were 5, 20 and 10 μg mL-1 for NLB, NLX and NLT, respectively. Good correlation coefficients were evaluated at 0.9997, 0.9995 and 0.9996, for the previously selected drugs, respectively. The % RSD was in an acceptable limit indicating high precision. Excellent separation and detection with acceptable results were achieved with respect to migration time, peak area and resolution. The obtained results were statistically evaluated and then compared with those obtained from other published methods. The electrophoretic method was validated in compliance with ICH guidelines.