A series of Microsoft Excel spreadsheets were developed to simulate the process of separation optimization under isocratic and simple gradient conditions. The optimization procedure is performed in a stepwise fashion using simple macros for an automatic application of this approach. The proposed optimization approach involves modeling of the peak shapes in order for the simulation of predicted chromatograms to be possible. The performance of the selected optimization procedure was tested by means of experiments performed on a reversed-phase column that produced Gaussian peaks for all solutes, providing benefits for computational simplicity. The separation optimization by Microsoft Excel especially with simple and easily implemented macros is a challenging pedagogical tool for advanced analytical chemistry courses. An illustrative video given in the Supporting Information supports a novice Excel practitioner in following the proposed separation optimization procedure.