Surface-active ionic liquid (SAIL) surfactants have attracted attention as promising alternatives to conventional surfactants because of their tailor-made and tunable properties. SAILs also address the limitations associated with conventional surfactants including toxicity and formation of unstable micelles. Here, we investigated the aggregation behavior of three biocompatible choline N-lauroyl-amino acid (NLAA)-based ILs with different amino acid side chains in aqueous solutions. The micellar behaviors of NLAA-ILs were investigated using surface tensiometry, conductometry, dynamic light scattering (DLS) and transmission electron microscopy (TEM). The critical micellar concentration (CMC) of the NLAA-ILs was found to be 2 to 4-fold lower compared with the conventional surfactant sodium dodecyl sulfate (SDS). The thermodynamic behavior confirmed that the micelle formation of NLAA-ILs was stable, spontaneous and entropy driven at room temperature. DLS and TEM studies revealed that the size and shape of the micelles depended on the presence of an N�hydrogen group in the head group of the anion. Choline N-lauroyl glycinate (ChoNLG) and dicholine N-lauroyl aspartate (Cho2NLA) were predominantly produced as unilamellar vesicles in water whereas choline N-lauroyl sarcosinate (ChoNLS) formed small spherical micelles. Importantly, SAIL ChoNLG showed lower toxicity toward mammalian cells compared with the analogous ILs or the conventional surfactant SDS and similar toxicity to the conventional surfactant Tween 80. SAIL ChoNLG was more efficient at forming hydrophobic ion pairs with the macromolecular drug heparin compared with SAIL ChoNLS. These results clearly suggest that the biocompatible NLAA-ILs represent promising potential substitutes for conventional surfactants in various biomedical applications. © 2020 Elsevier B.V.