The fieldwork of BIOAMBIENT.ES was developed from March 2009 to July 2010. BIOAMBIENT.ES is a human biomonitoring study of environmental pollutants performed in Spain at the national level. This article aims to show the tasks performed before starting the fieldwork to ensure the quality of the samples and consequently the quality of the results. A total of 1,936 whole blood, serum and first-morning urine samples and 604 hair samples were collected from workers who attended the annual occupational health examination in 38 centers in the Peninsula, Ceuta and the Canary Islands. Before the fieldwork was started, the optimal sampling material and sample shipment was identified and fieldworkers were trained in their tasks. Due to the planning and organization of the pre-analytical phase, only 1% of the collected samples had to be rejected due to problem with spills, conservation, etc. In addition, the analyses conducted showed no pre-analytical interferences.