The aim of this present study is to explore a rapid, selective, and sensitive HPLC method for cleaning validation for the analysis of levothyroxine sodium from production area. Chromatographic separation of levothyroxine sodium was achieved at 6.73 min by using 40: 60 volume of acetonitrile and deionized water that contain 0.05% of orthophosphoric acid mobile phase at a flow rate of 1.5 mL/min and detection UV wavelength of 225 nm. The method was validated according to USP Category I requirements. Linearity for detector response was observed in the concentration range of 0.0045–27ppm and coefficient of determination (r2) for calibration curve was found exactly 0.9999 (r2 > 0.99). Percent recovery was found from 83.15 to 83.80% ranging from 0.02265 to 006795mg of L-T4. Precision and intermediate precision showed that the % RSD of both were 2.80 and 0.98 respectively which were in the range of acceptable limit (< 10) for cleaning validation. Limit of detection (LOD) and limit of quantification (LOQ) were obtained at 0.002 and 0.006 ppm respectively. The HPLC method was successfully applied for analysis of levothyroxine sodium samples from different parts of equipment’s in production area.