Paper-based analytical devices (PADs) have been developed and used extensively in the last two decades due to their many advantages, including rapid results, portability, cost-effectiveness, and ease of fabrication and use. Moreover, their versatility allows them to be used for applications ranging from point-of-care diagnostics to environmental monitoring and food safety analysis. Despite these advantages, PADs must overcome limitations such as low sensitivity and resolution compared with the gold standards. This chapter discusses how pretreatment—including separation, filtration, and preconcentration—addresses these limitations by enhancing detection sensitivity in complex samples. Furthermore, the role of paper as a versatile substrate is discussed, followed by the operational principles, advantages, disadvantages, and applications of the key techniques.