The forward extraction (FE) and backward extraction (BE) of humanized IgG4 Fab fragments using HDEHP-isooctane and -corn oil reverse micelles (RMs) with/without a non-ionic (Brij 30) and counterionic (TOMAC) surfactant, respectively, was evaluated, and both solvents were equally effective. Interphase precipitate formation during FE caused Fab fragment loss; however, acetone precipitation with an ethanol wash step enabled true percentage extractions to be determined. Brij 30 and TOMAC influenced extraction; TOMAC mainly increased BE at pH 8 with Brij 30 in FE. The highest extraction yields were without Brij 30 and TOMAC for isooctane and with TOMAC for corn oil. Water content analysis showed RMs formed in both solvents with/without Brij 30 and TOMAC, confirming extraction feasibility.