A sensitive and selective High Performance Thin Layer Chromatographic (HPTLC), method was developed for the quantification of ambroxol HCl in human plasma. Six parameters from validation guidelines linearity, precision, Limit of Detection (LOD), Limit of Quantification (LOQ), selectivity, accuracy and 3 parameters from stability including extraction efficiency, Matrix factor, Benchtop stability, Freeze-thaw stability were determined. Extracted sample were spotted on 20 × 10 cm, layer thickness 0.2 mm, E-Merck, Darmstadt, Germany, aluminum backed silica gel 60 F254 Thin Layer Chromatography (TLC) plates. HPTLC mobile phase proportion used was Acetonitrile: Methanol: Triethylamine (8.2:1:0.8). Ambroxol HCl was separated at 0.6 Rf value. Ambroxol HCl gives linear response in concentration range 200-1000 ng/spot with r2 0.997. %RSD for precision is 1.7 and 1 for two concentration level. %RSD of accuracy were found to be 10.80, 14.89, 5.1% for three concentration level. LOD obtained is 0.0011 ng/spot and LOQ is 0.0033 ng/spot. Extraction efficiency of ambroxol HCl is 51.83% with 4.7% RSD; Coefficients of variance for Matrix factor are 2.1 and 0.35 ng/spot for two concentration level. Coefficients of variance for Benchtop stability are 1.7, 1, 1.3 and 0.9 for two concentration level. Coefficients of variance for Freeze-thaw stability are 2.0, 2.7, 0.35 and 1.1 for two concentration level. All %RSD values were found to be in acceptable limit as per guidelines for bioanalytical method development and validation. The method is simple; sensitive for HPTLC determination of ambroxol HCl in human plasma can be useful for quality control of ambroxol HCl.