Abstract: The objective of this study was to determine the effects of biotin and rumen-protected choline supplementation on milk production, milk composition, live weight change, milk choline and blood parameters in crossbred Holstein Friesian dairy cows. Twenty four Holstein Friesian crossbred lactating dairy cows, averaging 48±13 days in milk, 15.5±0.8 kg of milk and 380±19 kg body weight were blocked by milking days first and then stratified random balanced for milk yield and body weight into 3 groups of 8 cows. The 1st group (control) received approximately 6 kg of 21% CP concentrate. The 2nd group was fed the same basal diet as the control group and supplemented with 20 mg day^-1 of Biotin (BASF (Thai) Co., Ltd.) filled in a capsule and 20 g RPC day^-1 top-dressed (Reashure^®, Balchem Co., Ltd.) and the 3rd group was fed the same basal diet as the control group and supplemented with 20 mg day^-1 of biotin filled in a capsule and 40 g RPC day^-1 top-dressed. All cows also received ad libitum grass silage (Brachiaria ruziziensis; 55 days cutting age) had free access to clean water and were individually housed in a free-stall unit and individually fed according to treatments. The experiment lasted for 10 weeks with the 1st 2 weeks as the adjustment period followed by 8 weeks of measurement period Feed offered and left after eating of individual cow were collected on 2 consecutive days weekly and at the end of the experiment feed samples were pooled to make representative samples for proximate and detergent analyses. Daily milk yields were recorded. Milk sample and dry matter intake were collected in 2 consecutive days weekly. Live weights were recorded at the start and at the end of the experiment. Milk samples were taken on 56 days of the experiment and subjected to milk choline. Blood parameters were also analyzed. The results showed no statistical significant differences in intakes, live weight change, milk compositions and blood parameters (p>0.05) however, milk yield, 3.5% fat-corrected-milk yield, milk component yields and milk choline were increased by biotin and rumen-protected choline supplementation. It is recommended in the present study that the addition of 20 mg day^-1 biotin and 20 g day^-1 rumen-protected choline could be beneficial to lactating dairy cows in early lactation.