Leaf gas exchange measurements provide an important tool for inferring a plant’s photosynthetic biochemistry. In most cases, the responses of photosynthetic CO 2 assimilation to variable intercellular CO 2 concentrations ( A/ Ci response curves) are used to model the maximum rate of carboxylation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, V cmax) and the rate of electron transport at a given photosynthetically active radiation (PAR; J PAR). The standard Farquhar-Von Caemmerer-Berry model is typically used with default parameters of Rubisco kinetic values and mesophyll conductance to CO 2 ( g m) derived from tobacco that impairs analytical reliability across species. To study this, here we measured the temperature responses of key in vitro Rubisco catalytic properties and g m in cotton ( Gossypium hirsutum cv. Sicot 71) and derived V cmax and J 2000 ( J at 2000 µmol m -2 s -1 PAR) from cotton A/ Ci curves incrementally measured at 15°C to 40°C using cotton and tobacco parameters with our new automated fitting R package ‘OptiFitACi’. When applied to cotton, the tobacco parameters produced unrealistic J 2000: V cmax ratio of <1 at 25°C, two- to three-fold higher estimates of V cmax, approximately 50% higher estimates of J 2000 and more variable estimates of V cmax and J 2000, compared to model parameterisation with cotton-derived values. We determined that errors arise when using a g m of 0.23 mol m -2 s -1 bar -1 or below and Rubisco CO 2-affinities under ambient O 2 ( K C 21%O2) outside 461 µbar to 627 µbar to model A/ C i responses in cotton. We show how the multi- A/ C i modelling capabilities of ‘OptiFitACi’ serves as a robust, user-friendly extension of ‘plantecophys’ by providing simplified temperature-sensitivity and species-specificity parameterisation capabilities to enable higher accuracy estimates of V cmax and J 2000.