AbstractCastration is a painful procedure routinely performed on piglets. Specific periods relative to castration and time sampling rules are used widely to quantify deviations in piglet’s behavior associated with castration rather than assessing behavior for the entire trial period. However, very limited work has evaluated time sampling recording rules to quantify behavioral changes to piglets undergoing castration. Therefore, the objective of this study was to validate the accuracy of six continuous time sampling periods (5, 10, 15, 20, 30, and 45 min), to quantify piglet behavior post-castration when compared with a full 60-min observation period. Sixteen Yorkshire-Landrace x Duroc piglets were surgically castrated. Data were collected using continuous observation (recording rule) of each individual male piglet per litter for 60-min post-castration. The 60-min continuous behavioral data set was then subdivided into six data subsets for each defined continuous time sampling period (5, 10, 15, 20, 30, and 45 min). Data from each continuous time sampling period and the full period data were analyzed using both a generalized linear mixed model and linear regression analysis. For the generalized linear mixed model, 30- and 45-min continuous time sampling periods were not different (P > 0.05) when compared with the full observation period data for all behaviors. For the linear regression analysis, affiliative interaction, sitting, walking, huddled up, prostrated, scratching, spasms, and trembling behaviors met the pairwise comparison accuracy criteria: [1) the coefficient of determination (R2) was > 0.90, 2) the intercept did not differ from 0 (P > 0.05), and 3) the slope did not differ from 1 (P > 0.05)] at the 45-min continuous time sampling period compared to full observation period. Results from this study suggest that a 45-min continuous time sampling period would be necessary to accurately investigate piglet behavior during the acute pain sensitivity time post-castration when considering both maintenance and pain-associated behaviors.