With an ever-growing market and continual financial pressures associated with the prohibition of antibiotic growth promoters, the poultry industry has had to rapidly develop non-antibiotic alternatives to increase production yields. A possible alternative is yeast and its derivatives, such as the yeast cell wall (YCW), which have been proposed to confer a number of beneficial effects on the host animal. Here, the effect of YCW supplementation on the broiler chicken was investigated using a quantitative proteomic strategy, whereby serum was obtained from three groups of broilers fed with distinct YCW feed supplements or a control basal diet. Development of a novel reagent enabled application of Proteominer™ technology for sample preparation and subsequent comparative quantitative proteomic analysis revealed proteins which showed a significant change in abundance (n = 167 individual proteins; p <0.05); as well as proteins which were uniquely identified (n = 52) in, or absent (n = 37) from, YCW-fed treatment groups versus controls. An average of 7.1% of proteins showed changes in abundance with YCW supplementation. A number of effects of these YCW supplements including immunostimulation (via elevated complement protein detection), potential alterations in the oxidative status of the animal (e.g., glutathione peroxidase & catalase), stimulation of metabolic processes (e.g., differential abundance of glyceraldehyde-3-phosphate dehydrogenase), as well as evidence of a possible hepatoprotective effect (attenuated levels of serum α-glutathione s-transferase) by one YCW feed supplement, were observed. It is proposed that specific protein detection may be indicative of YCW efficacy to stimulate broiler immune status, i.e. may be biomarkers of YCW efficacy. In summary, this work has developed a novel technology for the preparation of high dynamic range proteomic samples for LC-MS/MS analysis, is part of the growing area of livestock proteomics and, importantly, provides evidential support for beneficial effects that YCW supplementation has on the broiler chicken.