The use of the field emission gun in scanning electron microscopy permits the imaging of submicrometer-size features. However, achieving sub-micrometer analytical spatial resolution in electron probe microanalysis (EPMA) requires both reducing the electron beam size and reducing the accelerating voltage to achieve the desired sub-micrometer interaction volume. The resulting quantification of the first-row transition metals at low accelerating voltage, i.e., below 7–8 kV, is problematic as the main characteristic X-ray lines (Kα) cannot be excited at these conditions. Furthermore, the use of the Lα and Lβ soft X-ray lines for quantification is complicated by bonding and self-absorption effects resulting in not-yet-determined mass absorption coefficients and hence in the failure of the traditional matrix correction procedure. We propose two methods to circumvent these low-kilovolt (low-kV) analysis limitations: using the non-traditional FeLℓ line and using universal calibration curves for the more traditional FeLα and Lβ lines. These methods were successfully applied to Fe-sulfide minerals showing accurate quantification results by EPMA at reduced kV, necessary for accurate quantification of sub-micrometer sulfide grains.