Coenzyme Q10 is biosynthesized by living organisms and considered a medically necessary health constituent thereby drawing the attention of the market. Naturally‐producing microbes are commonly used for the commercial production of coenzyme Q10. The detection of extracted coenzyme Q10 is usually done by high‐performance liquid chromatography and mass spectrometry methods which require sophisticated instrumentation and trained personnel. However, routine quality assessment is required rapidly at multiple steps during the large‐scale production process which necessitates the development of a rapid and relatively easy method for coenzyme Q10 detection. In the present study, a thin‐layer chromatography method is developed for the rapid detection of coenzyme Q10 extracted from the natural producer Agrobacterium tumefaciens. Various mobile phases were screened for the thin‐layer chromatography of coenzyme Q10 and the optimum separation‐detection was observed with methanol and ethyl acetate (8:2, v/v). The same extracted coenzyme Q10 was also analyzed by high‐performance liquid chromatography (the standard method of detection), showing a similar profile as with commercially available standard coenzyme Q10. Furthermore, extract from unstained thin‐layer chromatography spot showed the characteristic spectrum (ultraviolet‐visible) as with standard coenzyme Q10. Conclusively, a simple and inexpensive method for the rapid detection of coenzyme Q10 was developed to aid in the commercial production of this medically important coenzyme.