Liquid chromatography–tandem mass spectrometry (LC-MSMS) is a powerful technique for the quantification of small molecules, proteins, and peptides, but some sample preparation (prep) is necessary prior to instrument analysis. With so many options available, choosing the optimal sample clean-up method can be complex. Understanding the principles of sample prep—combined with a structured approach to selecting and optimizing a technique—can pay dividends in time saved and more precise and robust LC-MSMS assays. This chapter reviews general principles of sample prep, discusses a wide range of sample prep techniques for small molecules, and provides guidance for choosing between sample prep techniques. Sample prep techniques for proteins/peptides and dried blood spots (DBS) are also addressed.