Validamycin is the most active component of the antibiotic and antifungal validamycin complex. Since it is widely used to protect rice crops, its persistence in soil and rice matrices may affect the quality and the health of the consumer. Methods for its estimation from soil matrices are not available. Besides, its analysis from complex matrices including grain and paddy husk is challenging. So very few reliable analytical methods are available for its residue analysis. Thus, we aimed to develop a new QuPPe technique and new HPLC-ESI-MS/MS analytical method for the estimation of validamycin in grain, paddy husk, and soil. The QuPPe method was validated with a linearity range of 0.00101 to 0.10134 μg/mL. The LOQ of validamycin in grain, paddy husk, and soil was 1.01013 μg/kg and the retention time was 5.62 min. These results will be helpful in further analysis of food safety and environmental pollution.