•Fundamentals of extraction and clean-up methods are given.•Application of extraction and clean-up methods for different alkaloids is provided.•Factors that influence each technique are discussed.•Advantages and limitations of each technique are addressed. Alkaloids are a chemically diverse group of natural products known for their pharmacological and toxicological effects. Plants are the best known source of these compounds. Because of their high complexity, herbal matrices must be properly processed in order to access alkaloid content, overcoming different analytical steps, such as sampling, sample preparation, isolation, quantification and data analysis. Notably, sample preparation just recently is calling the attention, being considered a bottleneck of analytical processes. In this review, we provide the current state of the art in sample preparation for alkaloids analysis in herbal matrices, focusing on both extraction and clean-up steps. Both classic and modern techniques, such as Negative-Pressure Cavitation Extraction (NPCE), Microwave Assisted Extraction (MAE), as well as microfluidic chips are addressed, including the technique fundamentals, factors to be optimized, and their applications. A general discussion is also given, pointing out the advantages and limitations of each technique.