Serum separation gels are problematic in therapeutic drug monitoring because they adsorb some of the target drugs; however, the adsorptive properties of drugs that cause clinical intoxication remain unelucidated. Drug adsorption to separators results in a decrease in the observed blood levels, which may lead to uncertain assessments of clinical toxicology. Therefore, this study aimed to clarify the effects of four brands of blood collection tubes with serum separation gels that are used in Japan on the blood concentrations of central nervous system-acting drugs. Amitriptyline-, amoxapine-, mirtazapine-, chlorpromazine-, and flunitrazepam-spiked plasma at respective intoxicated concentrations were incubated in blood collecting tubes with serum separators, namely Vacutainer, Neotube, Insepack, and Venoject, at 4°C or 25°C for up to 72 or 168 hr and compared with control tubes without a serum separator. The amitriptyline, chlorpromazine, and flunitrazepam concentrations significantly decreased, even in control tubes. All the tubes containing serum separators significantly reduced the observed drug concentration when incubated at 25°C, which was estimated using a power function. Except amoxapine, rest all drugs when incubated at 4°C showed a decrease in concentration, albeit to a lesser degree than at 25°C. The blood collection tube with the greatest decrease in concentration was the Vacutainer. In conclusion, although the possibility of drug degradation in plasma must be considered, control tubes are strongly recommended for clinical toxicology, in addition to therapeutic drug monitoring, because drug adsorption is less likely to occur in these tubes.