Raptors are highly susceptible to poisoning by anticholinesterase drugs and pesticides, such as organophosphates and carbamates, due to their carnivorous and/or insectivorous diets. Measurement of plasma cholinesterase inhibition is a proxy indicator of exposure to these agents; however, prior knowledge of the normal plasma cholinesterase activity of healthy birds is required for interpretation of results. Such normal values have not yet been established for most species of neotropical raptors. We collected blood samples from 133 raptors, including eleven species of Falconiformes (n  =  45) and seven species of Strigiformes (n  =  88). The birds had no clinical signs or history of possible exposure to anticholinesterase drugs and were housed in rehabilitation centers (free-ranging animals) and zoos (captive animals) in the state of São Paulo, southeastern Brazil. We found that plasma cholinesterase activity, as measured by the Ellman method, had a logarithmic distribution with a median of 0.58 µmols/mL for Falconiformes (quartiles: Q1  =  0.43; Q₃  =  0.73) and 0.96 µmols/mL for Strigiformes (Q₁  =  0.78; Q₃  =  1.25). There were no significant differences between sex or age classes within Falconiformes or Strigiformes. Captive Strigiformes in zoos had lower plasma cholinesterase activity values than birds housed in the rehabilitation centers, a pattern that did not occur in Falconiformes. These results provide reference values of plasma cholinesterase activity for the future interpretation of suspected pesticide poisoning in neotropical raptors.