Natural bioactive derivatives of camptothecin (CPT), 10-methoxycamptothecin (MCPT) and 10-hydroxycamptothecin (HCPT) have been confirmed to possess high antitumor activities. MCPT could be metabolized to HCPT in vivo. The HPLC method for the quantification of MCPT and HCPT was established and validated, and the pharmacokinetics and the tissue distribution of MCPT in rats after i.v. administration have been well carried out in our previous studies. To improve the further understanding of the in vivo behavior of MCPT, a rapid and sensitive UPLC–MS/MS method was developed and validated for the quantification of MCPT and HCPT in plasma and tissue samples, and the pharmacokinetics and tissue distribution as well as the bioavailability of MCPT after i.g. were also illustrated. The results showed that MCPT could be highly converted to its active metabolite HCPT in plasma with the AUC0-∞ value of (185.28 ± 61.73) ng h/mL and (717.25 ± 165.67) ng h/mL for MCPT and HCPT, respectively. Meanwhile, MCPT and HCPT were rapidly absorbed and diffused into all the tested tissues (heart, liver, spleen, lung, kidney and brain) after i.g. administration. Similar with the results after i.v. administration of MCPT, MCPT concentration in lung tissue was also extremely higher than in other tested tissues, which implied that MCPT might have a great potential for the treatment of lung cancer.