In this study, we aimed to validate existing plasma assays to measure biomarkers for maternal signalling in milk and saliva of lactating sows. These biological samples are minimally invasive to the animal and could give a physiological profile of maternal qualities available to their piglets. Sows were farrowed in a zero-confinement system, and their colostrum and milk samples were manually collected during naturally occurring let-downs (i.e. not induced) over the lactation period. Saliva sampling involved sows voluntarily accepting cotton buds to chew without restraint. Commercial kits designed for blood plasma were tested, and any modifications and results are given. We successfully measured total protein, cortisol, tumour necrosis factor-α (TNF-α) and oxytocin in pig milk and saliva and immunoglobulin G (IgG) in pig milk samples. We were unsuccessful at measuring relaxin and serotonin in these biological samples. We observed higher levels of biomarkers in milk than in saliva. The measurement of TNF-α in pig milk for the first time revealed increased levels with larger litters. This development will allow more detailed understanding of biomarkers in milk. There was also evidence that the minimally invasive technique of using saliva sampling did not interrupt natural oxytocin production around parturition.