Pharmaceutical analysis plays an important role in all steps of drug development processes. Analysis of active pharmaceutical ingredients in biological samples needs sample preparation techniques to prevent the signal of the analyte from interferences coming from matrix components. Ultrafiltration is a well-known technique used in the food and pharmaceutical industry. Commercial ultrafiltration devices have been frequently used on proteomics and metabolomics studies for sample preparation. In pharmaceutical analysis, these devices have been employed to analyze the free concentration of drugs in biological fluids after filtration. However, they have been rarely used to determine the total concentration of targeted compounds when it is compared with some other common sample preparation techniques. Ultrafiltration-based sample preparation might be used to clean-up the sample easily from matrix components especially on bioanalysis performed with high-performance liquid chromatography (HPLC). In the case of using protein precipitation agents on filtration procedure, the quantitative recovery of this non-selective unique technique is competitive with solid-phase extraction.