Potential new pharmacotherapeutics for smoking cessation therapy can be identified in simple neurobehavioral assays using zebrafish larvae. Recording the activity of neural networks or individual neurons in individual larvae could ultimately lead to a better understanding of underlying neuronal mechanisms of drug action thus augmenting drug discovery. Certain adjustments of nicotine dosage and application are required as behavioral experiments on freely swimming larvae are translated into experiments that record nicotine-induced changes of neuronal activity in agarose-embedded larvae. Embedding larvae does not seem to change the overall response to nicotine. However, an increase in locomotor activity occurs with a 10 to 15 min delay after addition of nicotine to embedded larvae compared to the immediate onset in freely swimming larvae. Higher dosage of nicotine used in short behavioral assays of freely swimming larvae, is not suitable for monitoring neural activity in embedded larvae for several hours as nicotine appears to cause irreversible changes in locomotor activity. Lower nicotine dosage allows repeated application of the drug that alternates with “rinse” phases during which locomotor activity can recover. Initial experiments demonstrate that chemicals that reduce the nicotine-induced locomotor activity in freely swimming larvae also reduce recorded muscle activity in embedded larvae. Focal application of chemicals to the head region or injection into the brain can provide further evidence of the specific action of a drug. Establishing nicotine-response assays for embedded zebrafish larvae represents the first step towards measuring nicotine-induced neuroadaptations of larval brain activity using methods such as calcium-imaging.