Live-capture and translocation are methods to deal with nuisance eastern gray squirrels in North America, but it is unknown how these methods affect squirrel physiology or survival. In this study we validated an enzyme immunoassay (EIA) to measure fecal cortisol metabolites (FCMs) in gray squirrels; assessed their stress response relative to the positioning of the live trap (sun, shade, or control); and assessed the impact of translocation on their long-term stress, movement patterns, and survival using FCM levels, body mass changes, and radiotelemetry. We found that a 5α-pregnane-3β,11β,21-triol-20-one EIA reliably detected acute stress in gray squirrel feces 12–24 h after the stressor; live traps positioned in the sun resulted in higher peak FCM levels compared with traps positioned in the shade; translocated squirrels experienced a 10% mortality rate, compared with no mortality in the controls, although overall fates were the same; translocated squirrels initially explored more and dispersed farther than controls, but after 2 weeks made similar movements; and after controlling for the effect of season, translocation did not affect long-term FCM levels or body mass; this conclusion must be tempered by the low number of recaptures. Our study demonstrates the utility of the FCM assay; that gray squirrels are extremely sensitive to capture, handling, and confinement; and that live-capture must be done in a way that minimizes exposure to additive environmental stressors.