Chlorinated paraffins (CPs) are high production chemicals, which leads to their ubiquitous presence in the environment. To date, few studies have measured CPs in humans and typically at relatively low concentrations, despite indications that exposure may be high compared to various persistent organic pollutants. The aim of this study is to investigate the in vitro biotransformation of CPs by human liver fractions. We determined the changes of the CP concentrations after the enzymatic transformation with human liver microsomes using a two-tiered in vitro approach. CP concentrations decreased with human liver microsomes, with the decreases of 33-94% after incubating with different groups of enzymes for 2 h. The profiles of CP rapidly shifted after the incubation with human liver microsomes. In addition, the concentrations of CPs and the biotransformation products were tentatively measured using high-resolution mass spectrometric analysis, including very short CP (carbon chain length <10), alcohols, ketones, and carboxylic acids. C‒C bond cleavage is a potential transformation pathway for CPs, and ketones are potential products of CP biotransformation, especially for long-chain CPs (C>17). The ketone products may be investigated as CP exposure biomarker in biomonitoring studies.