Biofortification of crops with selenium (Se) is an effective strategy to increase the consumption of this micronutrient. Inductively coupled plasma mass spectrometry (ICP-MS) is one of the most used techniques in quantifying Se. However, due to some disadvantages (high cost, long analysis time, and being destructive), there is a need for new alternatives to Se quantification. This study aimed at establishing instrumental parameters for Se quantification, using two energy-dispersive X-ray fluorescence spectrometry (EDXRF) techniques (benchtop and handheld) in Se-biofortified broccoli seedlings and comparing it with ICP-MS. The results showed that the selection of proper filters for both EDXRF systems was crucial for determining Se and, when compared with ICP-MS, presented similar results for selenium-biofortified broccoli seedling treatments (BS50Se). In addition, the EDXRF techniques presented a Se limit of detection (LOD) at 0.6–0.9 mg kg⁻¹. This study demonstrates that EDXRF systems were successfully applied to concentration measurement of Se in biofortified samples and linked to low cost and shorter analysis time.