Background: Iatrogenic hypothyroidism may develop following radioiodine-I131 (RAI) treatment of hyperthyroid cats and can be diagnosed using the thyroid stimulating hormone (TSH) stimulation test. Objectives: To assess the effect of non-critical illness on TSH stimulation test results in euthyroid and RAI-treated cats. To assess the correlation of low total-thyroxine (tT4), low free-thyroxine (fT4) and high TSH concentrations with TSH stimulation test results. Animals: 33 euthyroid adult cats and 118 client-owned cats previously treated with RAI. Methods: tT4, fT4, and TSH were measured, and a TSH stimulation test was performed in all cats. Euthyroid control cats were divided into apparently healthy and non-critical illness groups. RAI treated cats were divided into RAI-hypothyroid (post-stimulation tT4 ≤1.5µg/dL), RAI-euthyroid (post stimulation tT4 ≥2.3µg/dL OR post stimulation tT4 1.5-2.3µg/dL and pre:post tT4 ratio >1.5) and RAI-equivocal (post stimulation tT4 1.5-2.3µg/dL and tT4 ratio <1.5) groups. Results: Non-critical illness did not significantly affect the TSH stimulation test results in euthyroid or RAI-treated cats. There were 21 cats in the RAI-equivocal group. Twenty-two (85%) RAI-hypothyroid cats (n=26) and 10/71 (14%) of RAI-euthyroid cats had high TSH (≥0.3ng/ml). Twenty-three (88%) RAI-hypothyroid cats had low fT4 (<0.70ng/dL). Of the 5 (7%) RAI-euthyroid cats with low fT4 only one also had high TSH. Only 5/26 (19%) RAI-hypothyroid cats had tT4 below the laboratory RI (<0.78µg/dL). Conclusions and Clinical Relevance: The veterinary-specific chemiluminescent fT4 immunoassay and canine-specific TSH immunoassay, may be used to aid in the diagnosis of iatrogenic feline hypothyroidism.