Pesticides are commonly used in agriculture to control pests and improve crop yields, but they pose significant risks to human health and the environment due to potential residues in biological matrices. This study presents an innovative ultrasonicator-aided cartridge-based extraction method specifically designed for detecting pesticides in simulated biological samples, utilizing ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). To address the need for reliable analytical techniques in forensic science, we developed a robust method for determining Tetramethrin (TMT) and Dimethoate (DMT) in simulated gastric fluid and urine samples using UPLC-MS/MS. While various methods have been employed to analyze biological materials with different solvents, salt combinations, and solid-phase extraction (SPE) sorbents, there is limited research focused on optimizing conditions for detecting TMT and DMT using ultra sonicator aided cartridge based extraction method by following design of experiments. Moreover, no previous studies have reported on their detection in simulated urine and gastric fluid. Our approach involved ultrasound-facilitated HLB cartridge-based solid-phase extraction (SPE) to effectively extract the pesticides across a concentration range of 2.5 to 160 ng/mL and showed high sensitivity, precision, and accuracy, meeting the validation criteria established by the Scientific Working Group of Toxicology (SWGTOX). Its practicality and efficiency make it a valuable tool for analyzing pyrethroid and organophosphorus pesticides in forensic matrices, due to its efficient, rapid and straightforward sample preparation process.