Due to a lack of analytical capability, there is little objective information on the number and identity of individual designer drugs being abused by various user sub-populations. Development of such methodologies would be a significant contribution in advancing the science and practice of forensic toxicology for this variable and potentially harmful class of drugs. The first task of the current project was to construct a designer drug master database, a high-resolution, high mass accuracy QTOF-MS/MS mass spectral library, along with a triple quadrupole (QqQ) triggered MRM database that included up to 10 transitions per compound for a minimum of 500 individual designer drugs and metabolites. The project’s second task involved comparison of dilute-and-shoot-, crash-and-shoot-, online and classical SPE-, and “QuEChERS“ (Quick, Easy, Cheap, Effective, Rugged, and Safe)-based extraction methods for reliable recovery of designer drugs from urine and whole blood. The third task involved the formal validation of LC-QTOF-MS and LC-QqQ-MS approaches for comprehensive screening/confirmation of just over 500 designer drugs, including analysis of blind spiked and authentic urine and whole blood specimens. An additional goal was to explore 2D-LC coupled to QTOF-MS for the separation and analysis of isomeric and structurally related designer drugs. This report concludes that the data and technologies developed from this project will enable crime laboratories to screen rapidly for and confirm the presence of hundreds of designer drugs in forensic toxicological specimens.