Humans are consistently exposed to a myriad of chemicals during their daily lives through various sources, including dietary intake and environmental pollutants . Exposomics represents the comprehensive analysis of all environmental exposures and associated influences on various health outcomes . To allow a full assessment of the totality of chemical exposures in human samples, sensitive, reliable, and robust analytical techniques, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) are required. Multiclass or next-generation human biomonitoring (HBM) methods have recently been developed to monitor a wide range of predetermined chemicals and are needed to fully address the complex nature of chemical exposure . In contrast, non-targeted analysis (NTA) is an emerging technique for exposomics using high-resolution mass spectrometry (HRMS)-based assays and allows the exploration of unknown chemicals in human samples . Furthermore, suspect screening methods are using large chemical databases and spectral libraries to prioritize detected features and annotate compound identities . Both targeted and non-targeted exposomic methods face significant challenges, such as interferences from complex biological samples , and ensuring repeatability and consistency across large sample sets is mandatory, especially when dealing with low-abundance analytes (typically at the pg/mL levels or lower) . Addressing these challenges in current exposome research requires smart optimization of the entire analytical workflow, including sample preparation protocols. Efficient sample pretreatment before instrumental analysis can reduce interferences, separate, and concentrate analytes in diverse matrices and various sample preparation techniques have been optimized to analyze the exposome. Liquid extraction (LE) and protein precipitation (PPT) are widely used sample preparation methods for exposomics due to their comparably wide chemical coverage . However, these preparation techniques are prone to preserve matrix interferences which can hamper overall method performance due to limited sample clean-up. Recently, solid-phase extraction (SPE) is getting more attention as a potential option for exposomic-type sample preparation and is discussed as a preferred approach for future large-scale applications, owing to its promising capacity to reduce matrix effects (ME), enhance sensitivity, and improve the method consistency for high-throughput studies . SPE has been frequently used for the purification and extraction of various analytes from diverse sample matrices for human biomonitoring including urine , blood , and various other biological matrices . However, a systematic development of an exposome-scale SPE method that is compatible with both targeted and non-targeted data acquisition strategies has rarely been performed. This challenge originates from the complexity of designing a method capable of balancing method robustness and a broad analyte spectrum. Most existing SPE methods focused on a relatively limited selection of targeted analytes to obtain reliable protocols without assessing the expected coverage for potential NTA due to the lack of certified reference standards, which restricts their utility in NTA. In this study, a targeted high-throughput sample pretreatment method using SPE cartridges was thoroughly optimized to assure the consistency of the whole workflow for pooled urine and plasma. Optimization steps included selecting SPE sorbents and sample buffers, optimizing washing and elution steps, and improving efficiency.