Sample preparation is an important step of any biomedical analysis. Development and validation of fast, reproducible and reliable sample preparation methods would be very helpful in increasing productivity. Except for a few direct injection methods, almost all biological samples should at least be diluted before any analysis. Sometimes dilution is not possible because of the low concentration of the target analyte in the sample, and alternative pretreatments, such as filtration, precipitation and sample clean up using different extraction methods, are needed. This review focuses on the recent achievements in the pretreatment of biological samples and investigates them in six categories (i.e., dilution, filtration/dialysis, precipitation, extraction [solid-phase extraction, liquid–liquid extraction], novel techniques [turbulent flow chromatography, immunoaffinity method, electromembrane extraction] and combined methods). Each category will be discussed according to its productivity rate and suitability for routine analysis, and the discussed methods will be compared according to the mentioned indices.