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Chemical warfare nerve agents (CWNAs) are potent and irreversible inhibitors of acetylcholinesterase (AChE). Oxime reactivators are an important part of the standard treatment for CWNA exposure as they can reactivate inhibited AChE. Evaluating the oxime candidates of interest in biological samples requires analytical detection methods and oxime reactivators as a class of compounds have historically been notoriously difficult to isolate, detect and analyze in an analytical laboratory, and there are currently no sensitive or robust analytical detection methods in the literature. The goal of this study was to develop reliable and robust novel extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods to detect and quantitate 2-PAM, HI-6, HLö-7, and MMB-4 in a human AChE knock-in, mouse carboxylesterase knock-out (KIKO) mouse in vivo model. This study identified an LC column that achieved retention for all four oxime compounds which is a major advancement over past oxime methods. A unique extraction and chromatographic method was developed for each oxime. The developed methods were sensitive down to 0.5 ng/mL for 2-PAM, 50 ng/mL for HI-6, and 15 ng/mL for both HLö-7 and MMB-4. These methods were validated to meet the Food and Drug Administration (FDA) bioanalytical method validation requirements under Good Laboratory Practice (GLP) conditions. The 4 methods were validated for performance by assessing linearity, sensitivity, precision, accuracy, selectivity, specificity, carryover, extraction recovery, dilution analysis, and stability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)