Purpose: The efficacy of two non-invasive tonometers, TonoLab and TonoPen XL, in detecting physiological or pharmacological changes of intraocular pressure (IOP) in mouse eyes, was assessed by comparison with a microneedle method. C57BL6 mice, bred under the 12-hr light and dark cycle over 2 weeks, were used. Under systemic anesthesia, mouse eyes were cannulated by a microneedle connected to a transducer and a water reservoir. We manipulated the intracameral pressure by changing the reservoir height, and obtained tonometer readings at each pressure (n = 39) with TonoLab and TonoPen XL. The correlation between each tonometer and the manometer was analyzed. Then the diurnal variation of IOP in the light and dark phases, and the IOP-lowering effect at 2 hr after latanoprost instillation, were measured with TonoLab, TonoPen XL, and a microneedle tonometer (n = 8). In mouse eyes, TonoPen XL could not show reliable scores, but TonoLab readings showed a strong correlation with manometer readings (y = 0.87x – 0.27, r² = 0.917). Nocturnal elevation of IOP in mouse eyes was significantly indicated with TonoLab and a microneedle tonometer (p < 0.001), but not with TonoPen XL. Latanoprost significantly reduced IOP by 2.1 ± 2.8 and 2.0 ± 1.0 mmHg with TonoLab and a microneedle tonometer, but not with TonoPen XL. TonoLab provides similar readings to a microneedle tonometer, and diurnal variation and drug effect were detectable in mouse eyes. TonoLab promises to be a non-invasive and useful method to evaluate physiological and pharmacological studies in mouse eyes.