It is widely accepted that the absence of suffering no longer defines animal welfare and that positive affective experiences are imperative. For example, laying hens may be housed in environments that do not cause chronic stress but may lack particular resources that promote positive affective experiences, such as conspecifics or effective enrichment. Despite a consensus of how important positive affect is for animal welfare, they are difficult to identify objectively. There is a need for valid and reliable indicators of positive affect. Pharmacological interventions can be an effective method to provide insight into affective states and can assist with the investigation of novel indicators such as associated biomarkers. We aimed to validate a pharmacological intervention that blocks the subjective hedonistic phase associated with reward in laying hens via the administration of the non-selective (μ, δ, and κ) opioid receptor antagonist, nalmafene. We hypothesized that nonfood deprived, hens that did not experience a positive affective state when presented with a mealworm food reward due to the administration of nalmefene, would show minimal anticipatory and consummatory behavior when the same food reward was later presented. Hens (n = 80) were allocated to treatment groups, receiving either nalmefene or vehicle (0.9% saline) once or twice daily, for four consecutive days. An anticipatory test (AT) was performed on all days 30 min post-drug administration. Behavioral responses during the appetitive and consummatory phase were assessed on days 1, 3 and 4. Anticipatory behavior did not differ between treatment groups the first time hens were provided with mealworm food rewards. However, antagonism of opioid receptors reduced anticipatory and consummatory behavior on days 3 and 4. Feed intake of standard layer mash was not impacted by treatment, thus nalmefene reduced non-homeostatic food consumption but not homeostatic consumption. Behavioral observations during the AT provided no evidence that nalmefene treated hens were fearful, sedated or nauseous. The results suggest that we successfully blocked the hedonistic subjective component of reward in laying hens and provide evidence that this method could be used to investigate how hens perceive their environment and identify associated novel indicators to assess hen welfare.