An optimized k0-standardized neutron activation analysis method incorporating cyclic irradiations (k0-CNAA) for short-lived radionuclides (SLRNs) has been developed at the Dalat research reactor. This paper highlights precise reactor parameter characterization using a cyclic irradiation system, simple sample preparation, and advanced calibration of HPGe detector-based gamma-ray spectrometry for SLRNs’ rapid multielement determination. By targeting SLRNs such as 77mSe, 110Ag, 20F, 179mHf, 52V, and 46mSc, with half-lives from seconds to minutes, the method enables quantification of elements essential for biological and environmental research. The in-house developed “k0-Dalat” software, featuring high automation, supports complete analysis. Method accuracy was validated using certified reference materials (SMELS-I, NIST-SRM-1566b, NIST-SRM-2711a), achieving deviations under 8% from certified values. Detection limits ranged from 0.1 to 1.9 mg/kg for target elements in biological samples, confirming the method’s high sensitivity and suitability for similar matrices.