The concepts of information theory were applied to the high-performance liquid chromatography (HPLC) technique, with diode-array (DAD) and/or fluorescence (FLD) detections. The information amount for a complete analysis can be computed as a function of analytical parameters, such as the number of analytes, level of concentration, and standard deviation of determinations. By means of the proposed method, the information content of a qualitative and quantitative analysis accomplished by HPLC-DAD-FLD was estimated, and sensitivity was optimized taking into account a maximum information content, while the detection limit was estimated considering that at this level of concentration the information content approaches zero.