A sensitive technique for speciation and quantification of Cr(III), Cr(VI), and total chromium has been developed using high-pressure liquid chromatography (HPLC) combined with direct injection nebulization (DIN) and inductively coupled plasma mass spectrometry (ICP-MS). A characterization and optimization of a microcolumn specifically designed for chromium speciation has been conducted. The direct injection nebulizer (DIN) was optimized to produce the best possible sensitivity for the system. Parameters affecting the column component of the system such as sample pH, eluent strength, sample size, flow, and pressure characteristics were studied in addition to optimal DIN-ICP-MS parameters. The detection limit obtained was 30, 60, and 180 ng/L for total chromium, Cr(III), and Cr(VI), respectively. Total chromium and speciated chromium were determined in one measurement with an analysis time of approximately 500 s. Accuracy measurements for the two chromium species were within 5% of the certified value for NIST Standard Reference Materials 2108 and 2109. In order to demonstrate the capability of the technique to analyze a true environmental sample matrix, industrial effluent samples were analyzed, and the results were compared with an alternative conventional method.