This study evaluates the toxicological and biochemical response of two-spotted spider mites to clofentezine selection pressure. The mortality rate of Tetranychus urticae in adult females depends on increased clofentezine concentration and clofentezine was found to be effective against females. The resistance rate of the CUM strain selected 12 times once per generation with clofentezine was increased from 1.28- to 105.27-fold. The interaction of some synergists with clofentezine was analyzed in the clofentezine-resistant CLOF 12 strain. Synergists had no effect on clofentezine toxicity. The clofentezine-resistant CLOF 12 strain showed resistance against chlorpyrifos, abamectin, propargite, fenpyroximate and amitraz. The modes of inheritance of resistance to clofentezine were found to be incompletely dominant and not sex-linked. Esterase enzyme activity was detected both by gel electrophoresis and microplate reader methods, while glutathione S-transferase (GST) and monooxygenase (P450) activity were detected only by the microplate reader method. During the selection period the esterase, the GST and the P450 enzymes activities were raised from 7.69, 7.09 and 0.003 3 to 18.40, 13.11 and 0.003 7 milli-optical density/min/mg proteins, respectively. An increase was observed in the band intensity of esterases and esterase enzymes may play a role in clofentezine resistance in T. urticae.