Fluid therapy selection is an important consideration for managing hemodynamically unstable patients. When working with reptiles, limited information is available for species-specific osmolality. A further understanding of reptile fluid balance and plasma osmolality is needed to select the most appropriate fluid for each situation. To date, osmolality has not been reported in a chameleon. Blood was collected from the ventral tail vein of 12 veiled chameleons (Chamaeleo calyptratus) and 12 panther chameleons (Furcifer pardalis) to measure plasma osmolality using an osmometer and to measure sodium, potassium, glucose, and uric acid concentrations using a standard chemistry analyzer. The chemistries were used to calculate osmolality using seven different formulas. The osmometer measured osmolalities in the veiled and panther chameleons were 330.5 mOsm/kg (range: 285–346.5 mOsm/kg) and 308.0 mOsm/kg (294–358.5 mOsm/kg), respectively. Bland–Altman plots were used to assess the level of agreement between these measured osmolalities and the seven calculated osmolalities. There was poor agreement between the measured and calculated osmolalities. Multiple linear regression was used to develop a species-specific equation for calculated osmolality; this was useful for veiled chameleons (osmolality = 2.098 [Na+]), but not panther chameleons. When comparing the measured osmolalities of these chameleons to other reptiles, there were significant differences (P < 0.05) between all groups, except veiled chameleons and green iguanas (Iguana iguana). The results of this study suggest that plasma osmolality should be considered species-specific in reptiles, and that an osmometer is the preferred method for measuring osmolality in veiled and panther chameleons.