A specific LC-MS/MS method was developed and validated for automated determination of codeine in human plasma, using on-line solid phase extraction (SPE) system coupled with positive ion electrospray ionization tandem mass spectrometry. The method allowed plasma direct injection onto cartridge without sample pre-treatment. Total analysis time per run was 3 min, allowing high-throughput for codeine determination. SPE on-line along a monolithic column (Chromolith Performance RP-18e, 100 mm x 4.6 mm) demonstrated to be highly effective in terms of backpressure, separation speed and peak asymmetry. Calibration curves range was linear 5.0-200 ng mL(-1). Method showed an excellent intra-day and inter-day precision ranged from 2.34 to 7.25% (CV%) as well as great intra-day and inter-day accuracy, ranging from 97.64 to 110% (RE%). SPE-LC-MS/MS method provided selectivity, accuracy, precision, fastness and high-throughput to assess codeine pharmacokinetics in human plasma samples.4012529