Untargeted analysis using liquid chromatography with tandem mass spectrometry (LC–MS/MS) chemical profiling has become a valuable tool for providing insight into the variations found in a sample set. However, the large data sets often generated, frequently yielding upwards of 20,000 features or more, can be overwhelming. Key elements in creating an experimental design are one of the most pivotal aspects of verifying statistical variance in a sample set. Therefore, care should be taken when an experimental design is developed and when statistical tools are used to perform data reduction and processing.