Recent improvements in chemical synthesis, compounds identification and bio-informatics have made new drug candidates increase exponentially, demanding that decisions about molecules’ potency are done with speed and accuracy. For that, pharmaceutical industry has used a combination of high-throughput screening (HTS) technologies based in pharmacological activity and pharmacokinetic (PK) properties of drug candidates. Some years ago, PK properties were the main reason for attrition in drug discovery and development. To overcome this, many HTS methodologies have emerged to optimize the screening phases and to help the selection of molecules with the best PK characteristics. Those methodologies are cost effective and efficient, preventing unnecessarily spends. Indeed, the primarily goal of pharmaceutical industries is to generate promise molecules based on efficacy, adequate safety and appropriated PK characteristics, avoiding failures in clinical trials. Bearing in mind that the majority of drugs are orally administered, besides solubility, as well the permeability becomes a key problem in gastrointestinal drug absorption. In this way, it is easy to understand the importance of the knowledge of potential permeability of drug candidates. Nowadays, for permeability screening, there are three important in vitro models that can be used: parallel artificial membrane permeability assay (PAMPA), Caco-2 cells assay and rat everted gut assay. This review presents these assays focusing on their advantages, limitations, experimental procedures and correlation between their permeability data. PAMPA, Caco-2 and rat everted gut should not be considered competing permeability assays, but complementary methods used in different phases of drug discovery.