Biomonitoring, or the measurement of environmental chemicals in human tissues and fluids, is used to supplement—and in some cases replace—more traditional exposure assessments which measure chemicals in environmental media. Volatile organic compounds (VOCs) in physiological fluids are biomarkers of exposure that present numerous challenges for sample collection and analysis. To date, a thorough evaluation of methods for collection and analysis of breast milk samples for volatiles has not been conducted. In this paper, we describe the development and validation of methods for collecting, storing, and analyzing 36 volatile organic compounds (VOCs) in breast milk to assess VOC exposure of lactating women and nursing infants. Volatile analyte loss was minimized by collecting and storing samples in containers with small headspace volume resulting in recovery ≥70% for all 10 VOCs detected in most breast milk samples. Potential contamination by chloroform, benzene, toluene, ethylbenzene, xylenes, and methyl-tert-butyl ether was minimized by using specially treated sample collection materials. Method detection limits in the low parts per trillion range were achieved by using solid-phase microextraction headspace sampling, gas chromatography, and selective ion monitoring mass spectrometry. We used this method to analyze 3 mL aliquots of breast milk collected from 12 women and found that 10 of the 36 VOCs were detectable in most samples (median values follow): m/p-xylene, 0.539 ng mL−1; toluene, 0.464 ng mL−1; 1,4-dichlorobenzene, 0.170 ng mL−1; tetrachloroethylene, 0.165 ng mL−1; o-xylene, 0.159 ng mL−1; ethylbenzene, 0.0149 ng mL−1; styrene, 0.129 ng mL−1; benzene, 0.080 ng mL−1; chloroform, 0.030 ng mL−1; and methyl-tert-butyl ether, 0.016 ng mL−1.