The aim of the study was to explore the possibility of assessing lean body mass (LBM) based on UV absorbance measurements in spent dialysate. 9 patients on chronic three-times-a-week HD (4 female, 5 male, mean age 58.8 ± 8.6 years) were studied. Blood and spent dialysate samples were collected for 3 consecutive hemodialysis (HD) sessions from every patient. A double-beam spectrophotometer was used for the determination of UV absorbance in the collected spent dialysate samples. Dual-energy x-ray absorptiometry (DXA) scans were performed on an interdialytic day. LBM was calculated based on creatinine concentration in blood (LBMblood) and UV absorbance in spent dialysate (LBMa) and assessed by DXA (LBMDXA). Also, in vitro experiments were carried out to investigate the effect of tissue hydration on DXA measurements. Although LBMa was slightly lower compared to LBMblood, the estimates based on UV absorbance in spent dialysate presented greater accuracy and precision compared to LBMDXA. The significant difference between LBMblood and LBMDXA was with high probability caused by the altered tissue hydration of HD patents. In summary, the results show that it is possible to asses LBM based on UV absorbance in spent dialysate.