Measurements of highly radioactive actinides such as Np, Pu, Am, or Cm in as-fabricated or irradiated nuclear fuels are crucial to improve the safety and performance of the nuclear fuel cycle, but also necessary to find solutions to the environmental impact of long-lived actinides in nuclear waste. Currently, the most common technique of measuring the quantity and distribution of actinides in nuclear fuel is electron probe microanalysis (EPMA). However, the accurate analysis of actinides by EPMA is accompanied by numerous obstacles such as interferences in the M-line X-ray region, absorption edges, and the lack of reference materials. Therefore, the measurement protocol of radioactive and irradiated samples requires further investigation and most importantly facilitation. Here, we establish a guideline for precise quantification of actinide elements (Th, U, Np, Pu, Am, and Cm) in fabricated or spent nuclear fuels. For this purpose, we have characterized actinide-bearing reference samples to visualize spectral interferences, list peak, and background positions and employ the standardless calibration curve method for the measurements of Am and Cm. This newly derived analytical protocol was successfully tested on an irradiated fuel sample and can be now implemented as an improved guideline for the quantification of actinides.