Although modern analytical methods developed for monitoring antibiotics in several biological matrices are easily available, none of them have been applied to evaluate the transfer of tiamulin into sow milk. Therefore, this work was intended to analyse the concentrations of tiamulin in milk samples collected from lactating sows during and after a treatment consisting of three consecutive intramuscular applications of the antibiotic. The second aim of this investigation was to determine tiamulin concentrations in serum samples obtained from suckling piglets ingesting milk contaminated with the compound. Ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) was used to quantify the analyte in both matrices. Our investigation proved tiamulin was transmitted into the milk of lactating sows. The mean concentration of the antibiotic among samples collected 3 h after administration was 1043 μg/L. The mean level of tiamulin on days 1 and 2 was 876 μg/L and 902 μg/L, respectively. The highest mean concentration of the antibiotic (1061 μg/L) was observed in samples collected on day 3. The mean concentration of the antibiotic in serum samples collected from 3-day-old piglets was 22.2 μg/L. The association between their body weight and serum tiamulin concentration was not statistically significant (p = 0.456).