A rapid and sensitive method for determination of cystatin C (CC) protein in human blood by means of high performance liquid chromatography (HPLC) was developed and validated. Estimated glomerular filtration rate (eGFR) has been calculated for the patients with chronic kidney disease (CKD). Ion pair liquid chromatography technique was utilized to separate CC along with UV detection. Calibration curve with excellent correlation (r 2 = 0.99) over the range from 0.75 to 20.50 mg/L of CC was accomplished. Limits of detection and quantifycation were 0.375 and 0.75 mg/L of CC, respectively. The recoveries were in the range of 93.6-102.3%. Intra-assay and inter-assay variabilities were 8.2 and 6.14%, respectively.