The objective of this study was to develop methodology for biopsying the rumen and colon from dairy calves during weaning. Six Holstein dairy bull calves (45 ± 1.5 kg birth weight) were ruminally cannulated during the second week of life and were fully weaned on d 42. Colon and rumen tissue samples were collected on d 28, 35, 42, 49, 56 and 84. Calves were not sedated but restrained in a chute for sampling. The lubricated distal tip of the endoscope (100 cm length, 9.8 mm diameter) was gradually inserted into the calf’s anus. Six (±12.6 mg) colon tissue samples were collected (30–40 cm from the calf’s anus) every sampling with endoscopic biopsy forceps (single use capture hot biopsy forceps), which were inserted through the instrument channel. The instrument channel was washed with double distilled water and 70% ethanol between calves as well as outside of the endoscope. Furthermore, the endoscope was introduced through the ruminal cannula to harvest ruminal papillae. Endoscopic biopsy of the rumen with endoscopic biopsy forceps (capture hot biopsy forceps and alligator jaws with a needle biopsy forceps) was unsuccessful (85% of the time) because the endoscopic biopsy forceps were unable to excise rumen papillae due to connective tissue. Thereafter, an Allis clamp was used to retrieve the blind sac through the ruminal cannula to perform direct tissue biopsying. Surgical scissors were used to perform tissue biopsying after exteriorization of the blind sac through the ruminal cannula. Colon and rumen samples were washed in PBS and 2 samples submerged in formalin solution and stored at room temperature for light histology analysis and 4 samples were stored in RNA stabilization fluid for molecular biology analysis. The mean RNA integrity number for the rumen papillae and colon samples were 8.9 ± 0.13 and 8.7 ± 0.09, respectively. In conclusion, endoscopic biopsying can be used for tissue harvest in the colon in young calves. However, it was found that collecting rumen tissue through retracting the rumen and taking samples with surgical scissors was more successful than using an endoscopic biopsy.