The carbon concentration in the carburized steels was measured by electron probe microanalysis (EPMA) for a range of soluted carbon content in austenite from 0.1 to 1.2 wt%. This study demonstrates the problems in carbon quantitative analysis using the existing calibration curve derived from pure iron (0.008 wt%C) and graphite (99.98 wt%C) as standard specimens. In order to derive an improved calibration curve, carbon homogenization treatment was performed to produce a uniform Kα intensity in selected standard samples (AISI 8620, AISI 4140, AISI 1065, AISI 52100 steel). The trend of detection intensity was identified according to the analysis condition, such as accelerating voltage (10, 15, 30 keV), and beam current (20, 50 nA). The appropriate analysis conditions (15 keV, 20 nA) were derived. When the carbon concentration depth profile of the carburized specimen was measured for a short carburizing time using the improved calibration curve, it proved to be a more reliable and accurate analysis method compared to the conventional analysis method.