In the last two decades, a large number of new drugs from several drug classes have appeared on the illicit drug market. While some of these compounds have meanwhile been scheduled as controlled substances, the majority of them are (still) sold as so-called ‘legal highs’, mostly via the Internet. At the time they appear on the market the metabolism of these drugs is generally unknown. Therefore, it must be studied in order to obtain data necessary for analytical method development as well as toxicological risk assessment. In vitro metabolism studies of new designer drugs can be performed for identification and structure elucidation of new designer drug metabolites or to assess the qualitative and quantitative involvement of certain enzymes in the metabolism of a particular drug. In this review, the value of the following enzyme preparations for in vitro metabolism studies of new designer drugs will be discussed: liver microsomes, recombinant cDNA-expressed enzymes, liver cytosol, S9 mix, and hepatocytes. This will cover the major metabolic enzymes: cytochrome P450 monooxygenases, flavin-monooxygenases, monoamine oxidases, UDP-glucuronyltransferases, sulfotransferases, and catechol-O-methyltransferases. Important analytical aspects such as the value of mass spectrometric techniques will also be covered. Copyright © 2011 John Wiley & Sons, Ltd.