•We evaluated pre-analytical conditions and two RT-qPCR assays for the FCV detection.•Addition of a viral transport medium increases FCV stability in the sample.•FCV-detection after cell culture is more sensitive than direct RT-qPCR from swabs.•A combination of different RT-qPCR assays increases sensitivity of FCV detection. Feline caliciviruses (FCVs) are non-enveloped RNA viruses that exhibit high genetic variation. Two reverse transcription quantitative polymerase chain reaction (RT-qPCR) FCV assays (S1 and S2) were evaluated using samples from 300 field cats. The direct detection of FCV in swabs and after propagation in cell culture, as well as the influence of storage conditions, was assessed. FCV-RNA detectability on dry swabs was similar after storage at either 4°C or −20°C, but viral burdens were maintained for a longer time period when viral transport medium was used. A total of 97 (32%) samples was considered FCV PCR-positive. Of these, 81% and 77% tested positive directly from swabs using S1 and S2, respectively; 84% and 81% tested positive after enrichment in cell culture, respectively. Combined detection by RT-PCR directly from swabs and after VI was most sensitive (up to 96%). Neither of the methods alone were able to detect all FCV-positive samples. In conclusion, clinical samples should be collected in viral transport medium, stored at ≤4°C and processed as soon as possible. The combination of cell culture with RT-qPCR or detection directly from swabs using a combination of different RT-qPCR assays is recommended to reach a high sensitivity of FCV detection.