The prescription of optimal hydration status in hemodialysis patients remains a much disputed topic in dialysis treatment. In particular, assessment of the patients optimal weight ("target weight") poses considerable difficulties. Multifrequency bioimpedance spectroscopy analysis (BIS) has been recommended as a non invasive, practical, and relatively non expensive method to determine hydration and nutritional status in patients on maintenance hemodialysis (HD). In the current study we used whole body BIS analysis for determination of body water (BW) compartments; total body water (TBW), extracellular water (ECW), and intracellular water (ICW) in 133 healthy adults, and in 227 hemodialysis patients with end-stage renal disease. BIS results were compared to anthropometric measurements. Our results showed strong correlation between TBW measured by BIS in control group in comparison to anthropometric calculation (p = 0.001). In HD patients we observed higher range of TBW, and TBW/ECW ratio (from 15.6 to 56.1 L and from 0.33 to 0.78), as measured by BIS at pre-HD, and also post-HD period (TBW ranged from 13.1 to 56.2 L, ECW/TBW ratio ranged from 0.33 to 1.27). The TBW BIS results did not correlate with anthropometric calculation. We postulate using of multi-frequency bioimpedance technique in precise determination of fluid compartments and in consequence in the assessment of "target weight" in hemodialysis population.