A new analytical method based on CZE coupled with tandem MS detection (CE-MS/MS) has been developed for the simultaneous determination of nine penicillins of human and veterinary use (nafcillin, dicloxacillin, cloxacillin, oxacillin, ampicillin, penicillin G, amoxicillin, penicillin V and piperacillin), whose levels are regulated by the EU Council regulation No. 2377/90 in animal edible tissues. CE separation conditions, sheath liquid composition and electrospray parameters have been carefully optimized to reach high sensitivity and precision. Separation was carried out in a 96 cm total length fused-silica capillary (50 microm id, 360 microm od) using 60 mM ammonium acetate at pH 6.0 as running buffer. Lomefloxacin was used as internal standard. MS/MS experiments were carried out using an IT as analyzer operating in multiple reaction monitoring mode to achieve the minimum number of identification points established by the 2002/657/EC European Decision. The use of an SPE procedure in two steps, combining Oasis hydrophilic-liphophilic balance and Alumina N cartridges provide a satisfactory preconcentration and clean up treatment for meat samples after extraction of the compounds with ACN. Only Oasis hydrophilic-liphophilic balance cartridges were necessary in water samples to obtain adequate recoveries. The method has been characterized for its use in meat and water samples, using matrix-matched calibrations. For chicken muscle samples, LODs between 8 and 12 microg/kg have been obtained, in all cases lower than the maximum residue limits permitted. For water samples LODs from 0.18 to 0.26 microg/L could ensure its satisfactory application in aquatic samples.