Abuse of synthetic drugs is widespread worldwide. Studies indicate that piperazine designer drugs act as substrates at dopaminergic and serotonergic receptors and/or transporters in the brain. This work aimed to investigate the cytotoxicity of N‐benzylpiperazine, 1‐(3‐trifluoromethylphenyl)piperazine, 1‐(4‐methoxyphenyl)piperazine and 1‐(3,4‐methylenedioxybenzyl)piperazine in the differentiated human neuroblastoma SH‐SY5Y cell line. Cytotoxicity was evaluated after 24 h incubations through the MTT reduction and neutral red uptake assays. Oxidative stress (reactive oxygen and nitrogen species production and glutathione content) and energetic (ATP content) parameters, as well as intracellular Ca2+, mitochondrial membrane potential, DNA damage (comet assay) and cell death mode were also evaluated. Complete cytotoxicity curves were obtained after 24 h incubations with each drug. A significant decrease in intracellular total glutathione content was noted for all the tested drugs. All drugs caused a significant increase of intracellular free Ca2+ levels, accompanied by mitochondrial hyperpolarization. However, ATP levels remained unchanged. The investigation of cell death mode revealed a predominance of early apoptotic cells. No genotoxicity was found in the comet assay. Among the tested drugs, 1‐(3‐trifluoromethylphenyl)piperazine was the most cytotoxic. Overall, piperazine designer drugs are potentially neurotoxic, supporting concerns on risks associated with the abuse of these drugs. Copyright © 2015 John Wiley & Sons, Ltd. Piperazine designer drugs act as substrates at dopaminergic and serotonergic receptors and/or transporters in the brain. This work aimed to investigate the cytotoxicity of N‐benzylpiperazine, 1‐(3‐trifluoromethylphenyl)piperazine, 1‐(4‐methoxyphenyl)piperazine and 1‐(3,4‐methylenedioxybenzyl)piperazine in the differentiated human neuroblastoma SH‐SY5Y cell line. Complete cytotoxicity curves were obtained after 24 h incubations with each drug. A significant decrease in intracellular total glutathione content was noted for the drugs. All drugs caused an increase of intracellular free Ca2+ levels, accompanied by mitochondrial hyperpolarization.