The utilization of cold active lipases in organic solvents proves an excellent approach for chiral synthesisand modification of fats and oil due to the inherent flexibility of lipases under low water conditions. Inorder to verify whether this lipase can function as a valuable synthetic catalyst, the mechanism con-cerning activation of the lid and interacting solvent residues in the presence of organic solvent must bewell understood. A new alkaline cold-adapted lipase, AMS8, from Pseudomonas fluorescens was studiedfor its structural adaptation and flexibility prior to its exposure to non-polar, polar aprotic and proticsolvents. Solvents such as ethanol, toluene, DMSO and 2-propanol showed to have good interactionswith active sites. Asparagine (Asn) and tyrosine (Tyr) were key residues attracted to solvents becausethey could form hydrogen bonds. Unlike in other solvents, Phe-18, Tyr-236 and Tyr-318 were predictedto have aromatic-aromatic side-chain interactions with toluene. Non-polar solvent also was found topossess highest energy binding compared to polar solvents. Due to this circumstance, the interaction oftoluene and AMS8 lipase was primarily based on hydrophobicity and molecular recognition. The molec-ular dynamic simulation showed that lid 2 (residues 148–167) was very flexible in toluene and Ca2+. Asa result, lid 2 moves away from the catalytic areas, leaving an opening for better substrate accessibilitywhich promotes protein activation. Only a single lid (lid 2) showed the movement following interactionswith toluene, although AMS8 lipase displayed double lids. The secondary conformation of AMS8 lipasethat was affected by toluene observed a reduction of helical strands and increased coil structure. Overall,this work shows that cold active lipase, AMS8 exhibits distinguish interfacial activation and stability inthe presence of polar and non-polar solvents.