Rose (Rosa hybrida L.) is one of the most important specialty cut flowers produced. Characterization of physiological variability in photosynthetic efficiencies, respiration rate and chlorophyll content is one of the greatest challenges in assessing rose net primary production of flowering, quality of cut flowers and other rose uses. Two modern rose varieties Floribunda and Hybrid Tea were used to optimize different methods for analysing physiological characteristics (photosynthesis rate, respiration rate and chlorophyll content). Many parameters were optimized, five different lights intensity were used (600, 900, 1200, 1500 and 2000 μmol•m •s −1 gave the highest photosynthesis rate. Five different measuring times were used (8 am, 10 am, 12 pm, 2 pm and 4 pm) and 12 pm was shown to be the optimum time for measuring photosynthesis rate. Among two different weather conditions (sunny day and cloudy day) sunny day was selected. For respiration rate two different measuring times (1 and 2 hours) after darkness were studied and 1 hour was chosen. Among three different times (10 am, 12 pm and 2 pm) for measuring chlorophyll content, 12 pm was selected. Using these optimized variables will allow researchers to collect robust and reproducible results to be obtained from different studies, and in turn lead to improved yields of horticultural plants.