Testosterone is one of the anabolic androgen steroids (AAS) that has been abused to improve higher athletic performance by enhancing muscle development and recovery. The purpose of this study was to assess basal level of serum total testosterone in male athletes and to compare it with physically active and sedentary males. The study sample was composed of 40 males divided in four groups of different physical activity level: two athlete groups (basketball and football), one physically active and one sedentary group. The subjects were assessed for the level of total testosterone by Chemiluminescent Immunoassay, controlling for biological parameters including age, body mass index (BMI), lean body mass (LBM), diurnal variations. LBM of basketball players (70.2 ± 1.9 kg) was higher (p < 0.05) than other groups. Basketball players showed a lower testosterone level (10.8 ± 1.0) compared with other groups (p < 0.05). The highest significant difference (p < 0.05) according to basal level of total testosterone was between basketball players and sedentary (16.7 ± 1.5). Decreased basal level of total testosterone of basketball players correlates with high LBM in respect to other physically active groups (p < 0.05). Future suggestion for WADA is to review the upper reference limit of basal level of total testosterone after the vigorous training periods for Basketball players.