Thiols have been of enduring interest for many years because of their role in biological and pharmacological processes. Monitoring of total thiols content is very important in order to understand their function in living organisms. This paper describes the spectrophotometric method for the determination of total thiols concentration in urine. The method is based on derivatization with 1-benzyl-2-chloropyridinium bromide and ultraviolet detection of S-pyridinium derivatives at 316 nm. The analytical recovery and RSD values for precision within the calibration range were from 95.7 to 102.9% and from 2.1 to 8.4%, respectively. The concentration of total thiols normalized against creatinine for 38 apparently healthy subjects (19 women and 19 men) occurred in the range 17.2-73.7 and 25.7-83.6 mmol/ mol creatinine, respectively. There was no difference in the urinary excretion of thiols in men and women, but there was a significant statistical correlation between urine total thiols and age in the studied group.