This article addresses sample collection protocols for monitoring and research of environmental chemicals in human milk. The process of milk synthesis and secretion and variations in contents of constituents that may impact measurement of environmental chemicals are presented. Possible sources of variation include parity, stage of lactation, method of sampling, maternal nutritional status, and dietary intake. General principles regarding how and when to collect milk samples are provided. For any previously unstudied environmental chemical in milk, all sources of variance must be assessed before a meaningful sampling protocol can be devised.