This study demonstrates the use of high-performance liquid chromatography (HPLC) equipped with evaporative light scattering detection (ELSD) for the direct detection and quantitation of iron (II) in ferrous sulfate tablets of 65 mg strength, whereas the corresponding United States Pharmacopoeia (USP) monograph is based on a titrimetric technique. After evaluating the effects of buffer concentration and pH, the separation of iron (II) was accomplished using a ZIC-HILIC^® peek column with a hydrophilic interaction chromatography (HILIC) mode gradient elution. Typical validation parameters were evaluated to assess the method’s quantitative performance for iron (II) which included specificity, accuracy, precision, linearity, stability, and limit of detection. This technique provides a unique and practical alternative method for the accurate quantitation of iron (II) as well as separation from iron (III). Additionally, the detection and separation for cobalt II and Manganese II were also accomplished using the same technique in the presence of Iron II and Iron III.