Triton™ X-100 is a mild, non-ionic surfactant widely used in the industry during the GMP processing of several pharmaceutical molecules. This is due to its low cost and diverse applications, such as aiding cell lysis and virus inactivation. However, Triton™ X-100 has been banned by the European Chemicals Agency (ECHA) since 2021 as per the Registration, Evaluation, Authorization and Restrictions of Chemicals (REACH) due to its environmental impact. Since then, several eco-friendly surfactants have been developed to replace Triton X-100 and are commercially available for GMP use, making the selection of a replacement a daunting task. This study assessed the impacts of four eco-friendly surfactants that are GMP-ready: (1) Deviron® C16, (2) Tergitol™ 15-S-9, (3) Ecosurf™ EH-9, and (4) Triton™ CG-110 on microbial lysis, protein release, and their capture during purification. The model protein used for the study was expressed in E. coli. The harvested cells were resuspended in lysis buffer with 0.1% surfactant and then lysed using cycles of high-pressure homogenization. The soluble protein was extracted from the cell lysate was clarified by removing the insoluble fraction, flocculating the DNA by adding polyethyleneimine to a 3% final concentration, and filtered through a 0.22 μm pore size filter. The total protein amount released during the cell lysis was measured with the Bradford assay to calculate the lysis efficiency for each surfactant. The therapeutic protein was captured using Anion Exchange Chromatography (AEX). The AEX elution fractions were analyzed by SDS-PAGE to determine the purity of the protein. We found that Triton™ CG-110 is the most suitable replacement among the tested eco-friendly surfactants, due to its similar protein extraction and capture purity values to Triton™ X-100.