Temperature shift (TS) to a hypothermic condition has been widely used during protein production processes that use Chinese hamster ovary (CHO) cells. The effect of temperature on cell growth, metabolites, protein titer and quality depends on cell line, product, and other bioreactor conditions. Due to the large numbers of experiments, which typically last 2–3 weeks each, limited systematic TS studies have been reported with multiple shift temperatures and steps at different times. Here, we systematically studied the effect of temperature on cell culture performance for the production of two monoclonal antibodies by industrial GS and DG44 CHO cell lines. Three 2–8 day short-duration methods were developed and validated for researching the effect of many different temperatures on CHO cell culture and quality attributes. We found that minor temperature differences (1–1.5 °C) affected cell culture performance. The kinetic parameters extracted from the short duration data were subsequently used to compute and predict cell culture performance in extended duration of 10–14 days with multiple TS conditions for both CHO cell lines. These short-duration culture methods with kinetic modeling tools may be used for effective TS optimization to achieve the best profiles for cell growth, metabolites, titer and quality attributes. Although only three short-duration methods were developed with two CHO cell lines, similar short-duration methods with kinetic modeling may be applied for different hosts, including both microbial and other mammalian cells.