Successful sample analysis depends on the availability of the instrumentation in the laboratory and on the selection of a suitable chromatographic method. First, an appropriate technique (separation mode) should be selected, depending on the properties of the analyte(s) to be determined; then the development and optimization of the separation method follows. This topic was treated in depth by Snyder et al. [1]. In this chapter, various aspects of the individual liquid chromatographic (LC) 216modes are examined and approaches for adjusting the experimental variables and optimization of separation conditions are discussed with special attention given to computer-assisted methods.