Abstract: Hair provides a suitable sample inassessing long-term changes in hypothalamic-pituitary-adrenocortical axis (HPA) system activity. The procedure used for hair analysis is therefore very important. To ensure that our procedure is reliable, liquid nitrogen was used to deepfreeze the samples before powdering them. Experimental trials used three treatment types: blank group (phosphate buffered saline (PBS)) (n=3), old tube group (n=10) and new tube group (n=10). The potential effects of PBS and tubes was then investigated. Additionally, both the relationship between the results of two samples of the same individuals (n=16) yielded by the same procedures but at different times, and the relationship between hair cortisol concentrations and the number of received intense aggressions (n=16) were also investigated. The results showed that the cortisol concentration of the blank group was 0 pg/mg; new tubes did not differ from old tubes（F_（1，19）=0.041, P=0.843，one-way ANOVA); and there was a high association between the results of two samples of the same individuals, yielded by same procedures operated at different times (r=0.893，P=0.000). Moreover, hair cortisol concentrations were moderately correlated with the number of received intense aggressions (r=0.591，P=0.008). Therefore, PBS and tubes did not affect the results significantly, therefore, our procedure was deemed reliable.