The epithelial sodium channel (ENaC) is crucial for sodium absorption in several epithelial tissues including lung and kidney. Its involvement in various renal and pulmonary disorders makes ENaC a potential drug target. High-throughput screening using the automated patch-clamp (APC) technique appears to be a promising approach to discover novel ENaC modulators with (patho-)physiological and therapeutic implications. The aim of this methodological study was to establish APC measurements of ENaC-mediated currents. First, we confirmed functional expression of ENaC in a HEK293 cell line stably transfected with human αβγ-ENaC using conventional manual whole-cell patch-clamp recordings. For APC measurements, a standard enzymatic cell-detachment procedure was used to prepare single cell suspensions. This resulted in a high success rate of APC recordings with amiloride inhibitable ENaC currents. Using a γ-inhibitory peptide and the small molecule ENaC activator S3969, we demonstrate that APC recordings could reveal inhibitory as well as stimulatory effects on ENaC. Interestingly, the enzymatic cell-detachment protocol resulted in partial proteolytic ENaC activation. The portion of proteolytically activated channels could be reduced by prolonged incubation of suspended cells in cell culture medium. This recovery protocol enhanced the relative stimulatory effect of chymotrypsin, a prototypical serine protease known to cause proteolytic ENaC activation. Thus, this protocol may be particularly useful for identifying novel ENaC activators mimicking proteolytic channel activation. In conclusion, we have established a high-throughput screening method for the identification of novel ENaC activators and inhibitors using APC.